Reversible protein multimers, methods for their production and use

ABSTRACT

Some aspects of this invention are based on the recognition that reversible protein multimers in which monomeric proteins are conjugated to a carrier molecule via chelation complex bonds are stable under physiological conditions and can be dissociated in a controlled manner under physiological, nontoxic conditions. Accordingly, such protein multimers are useful for a variety of in vitro, ex vivo, and in vivo application for research, diagnostics, and therapy. Some aspect of this invention provide reversible MHC protein multimers, and methods of using such multimers in the detection and/or isolation of specific T-cells or T-cell populations. Because reversible MHC multimers can efficiently be dissociated, the time of MHC binding to T-cell receptors, and, thus, T-cell receptor-mediated T-cell activation can be minimized. The use of reversible MHC multimers as provided herein, accordingly, allows for the detection and isolation of bona fide antigen-specific CD8+ T cells without inducing activation dependent cell death, including rare, therapeutically valuable T-cells expressing T-cell receptors binding tumor antigens with high affinity. Methods for the production and use of reversible multimers are also provided.

RELATED APPLICATION

This application claims the benefit under 35 U.S.C. §119(e) of U.S.provisional application Ser. No. 61/389,092, filed on Oct. 1, 2010, theentire disclosure of which is incorporated herein by reference.

BACKGROUND

Fluorescent MHC-peptide multimers, commonly referred to as tetramers arewidely used to detect, enumerate, analyze and sort antigen-specific CD8+T cells (1, 2). Monomeric MHC-peptide complexes are produced byrefolding of a MHC heavy and light chain in the presence of a peptide ofinterest and subsequently are biotinylated at a C-terminally addedbiotinylation sequence peptide (BSP, LHHILDAQKMVWNHR, SEQ ID NO: 1) thebiotin-transferase BirA. Fluorescent conjugates are obtained by reactionof biotinylated MHC-1-peptide monomers with phycoerythrin (PE) orallophycocyanine (APC) labeled streptavidin (2, 3). Although suchreagents generally perform well, they have shortcomings: 1) they areheterogeneous in terms of stoichiometry and configuration, whichcompromises stringent binding analysis (4). 2) The enzymaticbiotinylation is tedious, expensive and in the case of unstableMHC-peptide monomers causes degradation during the enzymatic reaction,which is performed at elevated temperatures (5). 3) For some studiesantigen-specific CD8+ T cells are isolated by cell sorting (e.g. FACS orMACS). Conventional multimers stably bind to cells and induce strong Tcell activation, which can induces death and result in loss of CD8+ Tcells, questioning that the surviving cells are representative for theoriginal populations (5-8). To overcome this, reversible multimer havebeen introduced, which contain low affinity biotin analogues and hencedissociate upon addition of free biotin. With these reagentssignificantly improved sorting and cloning efficiencies are obtained;however, they are costly and tend to be less stable (5, 8).

SUMMARY

Some aspects of this invention relate to reagents for reversiblestaining of cells. Some aspects of this invention provide stainingreagents that comprise a core structure, referred to herein as a carriermolecule, to which a plurality of monomeric proteins can reversibly bindto form a protein multimer. In some embodiments, the carrier moleculeitself is a detectable molecule, for example, a fluorophore. In someembodiments, the proteins that reversibly bind to the carrier moleculeare proteins that also specifically bind a molecular target, forexample, a specific cellular ligand or receptor, allowing for stainingof the molecular target with the protein multimer. Since the bond of theprotein and the carrier molecule is reversible, the protein multimercomplex can be dissolved, resulting in dissociation of the proteins fromthe carrier molecule and, thus, in re-monomerization of the boundproteins.

The protein multimers provided by some embodiments described herein areuseful, for example, for cell staining and cell isolation procedures.The multimers provided by some embodiments of this invention can bedissolved under non-toxic, physiological conditions, for example, byaddition of an agent interfering with the reversible binding of proteinand core structure of the multimer. Accordingly, one advantage of theinstantly provided protein multimers is that, in contrast to most otherstaining agents, they do not expose stained cells to the undesirableeffects of permanent staining. Dissolution of the multimer structureused for staining live cells avoids undesired biological effects ofcontinued association of cells with a dye or a multimeric proteinstructure, for example, continued activation of T-cell receptorsignaling in MHC-stained T-cells. Staining procedures using theinstantly described protein multimers, thus, allow for “minimallyinvasive” staining of cells, which is a requirement for the isolation ofnative cells that are sensitive to staining procedures, including, forexample, certain T-cell populations.

Some aspects of this invention provide methods and reagents for thegeneration of peptide-loaded MHC molecules, for example, ofpeptide-loaded MHC class II molecules. In some embodiments, methods forthe efficient production of molecularly defined, homogeneouslypeptide-loaded MHC class II molecules are provided. In some embodiments,MHC class II molecules are loaded with an antigenic peptide that isconjugated to a tag, for example, a tag allowing for peptide or proteinisolation and/or purification by chromatography, for example, byaffinity or ion exchange chromatography. In some embodiments, tagged MHCclass II binding peptides are provided. In some embodiments, the tag isan acidic tag, for example, a tag comprising a plurality of acidic aminoacid residues, or an acidic fluorophore, for example, an acidic cyaninedye. In some embodiments, the tag, for example, the acidic tag, isreversibly conjugated to the antigenic peptide of interest, for examplevia a cleavable linker. In some embodiments, the cleavable linker is aphotocleavable linker. In some embodiments, the cleavable linkercomprises a cleavage site for an enzyme, for example, a protease, or achemical.

Some aspects of the invention, including aspects and embodiments notmentioned in this summary are described in more detail elsewhere in thespecification and in the claims.

Some aspects of this invention provide protein multimers that include(a) a multivalent carrier molecule, and (b) a plurality of proteinsbound to the carrier molecule. In some embodiments, at least one of theproteins is conjugated to the carrier molecule via a non-covalent bondwith a dissociation constant 1 μM>K_(D)≧10 fM. In some embodiments, atleast one of the peptides or proteins is conjugated to the carriermolecule via a chelate complex bond.

Some aspects of this invention provide multivalent chelants thatcomprise a water-soluble carrier molecule and a plurality of chelantmoieties conjugated to the carrier molecule.

Some aspects of this invention provide multivalent chelants thatcomprise a carrier molecule or structure, and a plurality of chelantmoieties conjugated to the carrier molecule or structure, wherein thecarrier molecule or structure has a diameter of less than 0.1 nm, lessthan 0.2 nm, less than 0.25 nm, less than 0.3 nm, less than 0.4 nm, lessthan 0.5 nm, less than 0.6 nm, less than 0.7 nm, less than 0.75 nm, lessthan 0.8 nm, less than 0.9 nm, less than 1 nm, less than 1.1 nm, lessthan 1.2 nm, less than 1.3 nm, less than 1.4 nm, less than 1.5 nm, lessthan 1.6 nm, less than 1.7 nm, less than 1.8 nm, less than 1.9 nm, lessthan 2 nm, less than 2.5 nm, less than 3 nm, less than 4 nm, less than 5nm, less than 6 nm, less than 7 nm, less than 8 nm, less than 9 nm, orless than 10 nm. In some embodiments, the diameter of the carriermolecule is less than 20 nm, less than 30 nm, less than 40 nm, less than50 nm, less than 60 nm, less than 70 nm, less than 80 nm, less than 90nm, less than 100 nm, less than 200 nm, less than 300 nm, less than 400nm, less than 500 nm, less than 600 nm, less than 700 nm, less than 800nm, less than 900 nm, or less than 1 μm.

Some aspects of this invention provide methods for the production ofprotein multimers that comprise a step of contacting a monomeric chelantmoiety-conjugated MHC molecule with a carrier molecule, for example, awater-soluble carrier molecule or a carrier molecule or structuredescribed herein, that is conjugated to a plurality of chelant moietiesunder conditions suitable for formation of a chelate complex between thechelant moieties conjugated to the MHC molecule and the chelant moietiesconjugated to the carrier molecule.

Some aspects of this invention provide methods for the production of MHCmolecules that are conjugated to a ligand via a chelate complex bond,comprising a step of contacting an MHC molecule conjugated to a firstchelant with a ligand molecule conjugated to a second chelant underconditions suitable for formation of a chelate complex between the firstand the second chelant. In some embodiments, the resulting MHC moleculeis then contacted with a multivalent binding molecule binding the ligandto produce reversible MHC multimers.

Some aspects of this invention provide methods to generatepeptide-loaded MHC molecules that are conjugated to a chelant using MHCbinding peptides conjugated to a tag, the methods comprising the stepsof providing an MHC molecule bound to an antigenic MHC molecule-bindingpeptide that is conjugated to a tag via a cleavable linker, removing thetag from the antigenic peptide, and conjugating a chelant moiety to aheavy chain of the MHC molecule.

Some aspects of this invention provide methods for staining, detecting,and isolating cells, the methods comprising a step of contacting apopulation of cells with a protein multimer as described herein, forexample, with a multimer comprising a chelate bond and a detectablelabel, and performing an assay to detect a cell binding the multimer.

Some aspects of this invention provide method for the isolation of cellsthat bind a multimer as described herein, the methods comprising thesteps of (a) contacting a population of cells with a protein multimer asprovided herein, for example, with a multimer comprising a chelate bondand a detectable label, (b) optionally, detecting a cell binding themultimer, and (c) isolating the cell binding the multimer.

Some aspects of this invention provide methods for the manipulation ofT-cell populations with protein multimers, the methods comprising a stepof contacting a population of cells expressing a T-cell receptor with anMHC multimer as described herein under conditions suitable for themultimer to bind to the T-cell receptor and for a time sufficient forthe T-cell receptor/MHC class I molecule interaction to activate aT-cell expressing the T-cell receptor and binding the MHC multimer.

Some aspects of this invention provide cells or cell populations thatare contacted with a protein multimer as provided herein, for example,with an MHC multimer.

Some aspects of this invention provide kits comprising peptide-loaded orempty MHC molecules as provided herein.

Some aspects of this invention provide isolated peptide-loaded MHCmolecules that comprise an MHC heavy chain, and an antigenic peptide. Insome embodiments, the peptide is conjugated to a tag, for example, a tagfor ion exchange chromatography.

Some aspects of this invention provide methods for the generation ofpeptide-loaded MHC molecules, for example, MHC class II molecules,comprising a step of contacting an empty MHC molecule with an antigenicpeptide conjugated to a tag under conditions suitable for the antigenicpeptide to bind the MHC molecule. In some embodiments, the tag is a tagfor ion exchange chromatography and the method, in some embodiments,includes a step of isolating peptide-loaded MHC molecules by performinga chromatography procedure.

Some aspects of this invention provide methods for the generation of MHCclass II molecules, empty or peptide-loaded, the method comprising astep of contacting an MHC class II type alpha heavy chain with an MHCclass II type beta heavy chain under conditions suitable for the alphaand the beta chain to form a heterodimeric MHC class II molecule. Insome embodiments, at least one of the MHC class II heavy chains isconjugated to a tag. In some embodiments, a step of isolating the MHCclass II molecule is performed, wherein the isolating comprises a stepof chromatography, for example, affinity chromatography.

Some aspects of this invention provide a protein multimer comprising (a)a multivalent carrier molecule, and (b) a plurality of proteins bound tothe carrier molecule. In some embodiments, at least one of the pluralityof proteins is conjugated to the carrier molecule via a non-covalentbond with a dissociation constant 1 μM>KD≧10 fM. In some embodiments,the dissociation constant is KD≧0.1 pM. In some embodiments, thedissociation constant is KD≧1 pM. In some embodiments, the dissociationconstant is KD≧10 pM. In some embodiments, the dissociation constant isKD≧100 pM. In some embodiments, the dissociation constant is KD≧1 nM. Insome embodiments, the dissociation constant is KD≧10 nM. In someembodiments, the dissociation constant is KD≧100 nM. In someembodiments, the dissociation constant is KD<10 nM. In some embodiments,the dissociation constant is KD<1 nM. In some embodiments, thedissociation constant is KD<100 pM. In some embodiments, thedissociation constant is KD<10 pM. Some aspects of this inventionprovide a protein multimer comprising (a) a multivalent carriermolecule, and (b) a plurality of proteins bound to the carrier molecule.In some embodiments, at least one of the plurality of proteins isconjugated to the carrier molecule via a chelate complex bond. In someembodiments, the chelate complex bond is a bond with a dissociationconstant 5 μM>KD 1 fM. In some embodiments, the protein is an MHCmolecule and the protein multimer is an MHC multimer. In someembodiments, the chelate complex bond comprises a chelant conjugated tothe MHC molecule, and a chelant conjugated to the carrier molecule. Insome embodiments, the chelant conjugated to the carrier molecule is of adifferent structure than the chelant conjugated to the MHC molecule. Insome embodiments, the MHC molecule comprises an MHC a chain. In someembodiments, the MHC molecule further comprises an MHC α chain or a β2microglobulin chain. In some embodiments, the MHC molecule is an MHCclass I molecule. In some embodiments, the MHC molecule is an MHC classII molecule. In some embodiments, the chelant conjugated to the MHCmolecule is C-terminally conjugated to the MHC β chain. In someembodiments, the chelant conjugated to the MHC molecule is C-terminallyconjugated to the MHC β chain or the β2 microglobulin chain. In someembodiments, the chelant conjugated to the MHC molecule is a peptidecomprising a chelant moiety. In some embodiments, the peptide comprisinga chelant moiety is fused to a polypeptide chain comprised by the MHCmolecule. In some embodiments, the peptide comprising a chelant moietycomprises a poly-Histidine sequence. In some embodiments, thepoly-Histidine sequence comprises 3-24 His residues. In someembodiments, the chelant conjugated to the MHC molecule comprises a His6tag, a His12 tag, or a 2× His6 tag. In some embodiments, the chelantconjugated to the carrier molecule comprises an NTA moiety. In someembodiments, the NTA moiety is bound to the carrier molecule in mono-NTAconfiguration. In some embodiments, the NTA moiety is bound to thecarrier molecule in poly-NTA configuration. In some embodiments, the NTAmoiety is bound to the carrier molecule in di-NTA, or tetra-NTAconfiguration. In some embodiments, the NTA moiety is bound to a linker.In some embodiments, the linker comprises a maleimide moiety orderivative. In some embodiments, the linker comprises an oxime moiety orderivative. In some embodiments, the linker is between about 9 Å andabout 23 Å long. In some embodiments, the linker is covalently bound tothe carrier molecule. In some embodiments, the linker is covalentlybound to a ligand of a binding molecule, and wherein the bindingmolecule is covalently bound to the carrier molecule. In someembodiments, the ligand is biotin and the binding molecule isstreptavidin. In some embodiments, the chelate complex bond furthercomprises a divalent cation. In some embodiments, the divalent cation isan Ni2+, Cu2+, Zn2+, Co2+, Cd2+, Sr2+, Mn2+, Fe2+, Mg2+, Ca2+, or Ba2+ion. In some embodiments, the carrier molecule is a fluorophore, aphycobilin, phycoerythrin or allophycocyanine, a quantum dot (Qdot), amicrosphere (e.g., a fluorescent microsphere (e.g. Fluorospheres® type),a magnetic particle, or a nanoparticle. In some embodiments, the MHCmolecule is an empty MHC molecule or a peptide-loaded MHC molecule. Insome embodiments, the peptide-loaded MHC molecule is chosen from the MHCmolecules disclosed in Table 2. In some embodiments, the MHC moleculecomprises an HLA-A*0201 heavy chain. In some embodiments, the MHCmolecule is loaded with an antigenic peptide. In some embodiments, theMHC molecule is loaded with a peptide comprising the sequence GILGFVFTL(SEQ ID NO: 2). In some embodiments, the multimer is a tetramer. In someembodiments, the antigenic peptide is conjugated to a tag. In someembodiments, the tag is conjugated to the peptide via a cleavablelinker. In some embodiments, the linker is a photocleavable linker. Insome embodiments, the linker is an NPβA linker. In some embodiments, thelinker is a peptide linker that comprises an amino acid sequence thatcan be cleaved by a protease or by a chemical. In some embodiments, thetag is an acidic peptide tag. In some embodiments, the acidic peptidetag comprises a plurality of acidic amino acid sequences. In someembodiments, the tag is a pY-D4, pY-D5, pY-D6, pY-D7, pY-D8, pY-D9, orpY-D10 tag. In some embodiments, the tag is a pY-E4, pY-E5, pY-E6,pY-E7, pY-E8, pY-E9, or pY-E10 tag. In some embodiments, the tag is adesthiobiotin (DTB) tag.

Some aspects of this invention provide a multivalent chelant comprisinga water-soluble carrier molecule, and a plurality of chelant moietiesconjugated to the carrier molecule. Some aspects of this inventionprovide a multivalent chelant comprising a carrier molecule orstructure, and a plurality of chelant moieties conjugated to the carriermolecule or structure. In some embodiments, the carrier molecule orstructure has a diameter of less than 0.1 nm, less than 0.2 nm, lessthan 0.25 nm, less than 0.3 nm, less than 0.4 nm, less than 0.5 nm, lessthan 0.6 nm, less than 0.7 nm, less than 0.75 nm, less than 0.8 nm, lessthan 0.9 nm, less than 1 nm, less than 1.1 nm, less than 1.2 nm, lessthan 1.3 nm, less than 1.4 nm, less than 1.5 nm, less than 1.6 nm, lessthan 1.7 nm, less than 1.8 nm, less than 1.9 nm, less than 2 nm, lessthan 2.5 nm, less than 3 nm, less than 4 nm, less than 5 nm, less than 6nm, less than 7 nm, less than 8 nm, less than 9 nm, or less than 10 nm.In some embodiments, the diameter of the carrier molecule is less than20 nm, less than 30 nm, less than 40 nm, less than 50 nm, less than 60nm, less than 70 nm, less than 80 nm, less than 90 nm, less than 100 nm,less than 200 nm, less than 300 nm, less than 400 nm, less than 500 nm,less than 600 nm, less than 700 nm, less than 800 nm, less than 900 nm,or less than 1 μm. In some embodiments, the chelant moieties arenitrilotriacetic acid (NTA) moieties. In some embodiments, the NTAmoieties are in mono-NTA configuration. In some embodiments, the NTAmoieties are in poly-NTA configuration. In some embodiments, the NTAmoieties are in di-NTA, or tetra-NTA configuration. In some embodiments,the NTA moieties are bound to a linker. In some embodiments, the linkercomprises a maleimide moiety. In some embodiments, the linker comprisesan oxime moiety. In some embodiments, the linker is between about 9 Åand about 23 Å long. In some embodiments, the linker is covalently boundto the carrier molecule. In some embodiments, the linker is covalentlybound to a ligand of a binding molecule, and wherein the bindingmolecule is covalently bound to the carrier molecule. In someembodiments, the ligand is biotin and the binding molecule isstreptavidin. In some embodiments, the carrier molecule is afluorophore, a phycobilin, phycoerythrin or allophycocyanine, or aquantum dot (Qdot). In some embodiments, a plurality of the chelantmoieties conjugated to the carrier molecule form chelate complex bondsto a plurality of chelant moiety-conjugated monomeric molecules, thusforming a multimer of the monomeric molecule. In some embodiments, themonomeric molecule is a polyprotein. In some embodiments, the monomericmolecule is a small molecule compound. In some embodiments, themonomeric molecule is a polynucleotide. In some embodiments, themonomeric molecule is a ligand of a receptor. In some embodiments, thereceptor is a cell-surface receptor. In some embodiments, the receptoris a T-cell receptor. In some embodiments, the monomeric molecule is anMHC molecule. In some embodiments, the monomeric molecule is an MHCclass I molecule. In some embodiments, the MHC molecule comprises anHLA-A*0201 heavy chain. In some embodiments, the MHC molecule is an MHCclass II molecule. In some embodiments, the MHC molecule is loaded withan antigenic peptide. In some embodiments, the MHC molecule is loadedwith a peptide comprising the sequence GILGFVFTL (SEQ ID NO: 3). In someembodiments, the multivalent chelant is a tetravalent chelant. In someembodiments, the tag is an acidic peptide tag. In some embodiments, theacidic peptide tag comprises a plurality of acidic amino acid sequences.In some embodiments, the tag is a pY-D4, pY-D5, pY-D6, pY-D7, pY-D8,pY-D9, pY-D10, pY-E4, pY-E5, pY-E6, pY-E7, pY-E8, pY-E9, or pY-E10 tag.In some embodiments, the tag is a desthiobiotin (DTB) tag.

Some aspects of this invention provide a method comprising contacting amonomeric chelant moiety-conjugated MHC molecule with a water-solublecarrier molecule conjugated to a plurality of chelant moieties underconditions suitable for formation of a chelate complex between thechelant moieties conjugated to the MHC molecule and the chelant moietiesconjugated to the carrier molecule. In some embodiments, the chelantmoieties conjugated to the carrier molecule are NTA moieties. In someembodiments, the NTA moieties are in mono-NTA configuration. In someembodiments, the NTA moieties are in poly-NTA configuration. In someembodiments, the NTA moieties are in di-NTA, or tetra-NTA configuration.In some embodiments, the NTA moieties are bound to a linker. In someembodiments, the linker is between about 9 Å and about 23 Å long. Insome embodiments, the linker is covalently bound to the carriermolecule. In some embodiments, the linker is covalently bound to aligand of a binding molecule, and wherein the binding molecule iscovalently bound to the carrier molecule. In some embodiments, theligand is biotin and the binding molecule is streptavidin. In someembodiments, the carrier molecule is a fluorophore. In some embodiments,the carrier molecule is a phycobilin. In some embodiments, the carriermolecule is phycoerythrin or allophycocyanine. In some embodiments, thecarrier molecule is a quantum dot (Qdot). In some embodiments, thecarrier molecule is a magnetic particle. In some embodiments, thecarrier molecule is a nanoparticle. In some embodiments, the carriermolecule is PE conjugated to streptavidin. In some embodiments, thecarrier molecule conjugated to a plurality of chelant moieties isgenerated by incubating a carrier molecule conjugated to a plurality ofbinding molecules with an excess of chelant-conjugated ligand underconditions suitable for the ligand to bind the binding molecule. In someembodiments, the molar ratio of carrier:ligand is between 1:2 and 1:10.In some embodiments, the molar ratio of carrier:ligand is 1:5. In someembodiments, the incubating is performed at a temperature between 2-16°C. In some embodiments, the incubating is performed at about 4° C. Insome embodiments, the method comprises a step of incubating the carriermolecules contacted with the ligand with NiSO4. In some embodiments, themethod comprises a step of contacting the carrier molecule conjugated toa plurality of chelant moieties with a molar excess of the MHC moleculeconjugated to a chelant. In some embodiments, the excess is 2-20 fold.In some embodiments, the excess is 10-fold. In some embodiments, the MHCmolecule is an MHC class I molecule. In some embodiments, the MHCmolecule comprises an HLA-A*0201 heavy chain. In some embodiments, theMHC molecule is an MHC class II molecule. In some embodiments, the MHCmolecule is loaded with an antigenic peptide.

Some aspects of this invention provide a method comprising contacting anMHC molecule conjugated to a first chelant with a ligand moleculeconjugated to a second chelant under conditions suitable for formationof a chelate complex between the first and the second chelant. In someembodiments, the first chelant comprises a peptide. In some embodiments,the peptide is C-terminally fused to a polypeptide chain comprised bythe MHC molecule. In some embodiments, the peptide comprises apoly-Histidine sequence. In some embodiments, the poly-Histidinesequence comprises between 3 and 24 His residues. In some embodiments,the chelant conjugated to the MHC molecule comprises a His6 tag, a His12tag, or a 2× His6 tag. In some embodiments, the second chelant is NTA.In some embodiments, the NTA is in mono-NTA configuration. In someembodiments, the NTA is in poly-NTA configuration. In some embodiments,the NTA is in di-NTA, or tetra-NTA configuration. In some embodiments,the NTA is bound to a linker. In some embodiments, the linker iscovalently bound to the ligand molecule. In some embodiments, the linkeris between about 9 Å and about 23 Å long. In some embodiments, themethod further comprises contacting the ligand molecule with amultivalent binding molecule under conditions suitable for the ligand tobind the multivalent binding molecule. In some embodiments, the ligandis biotin and the binding molecule is streptavidin. In some embodiments,the binding molecule is conjugated to a carrier molecule. In someembodiments, the carrier molecule is a fluorophore. In some embodiments,the carrier molecule is a phycobilin. In some embodiments, the carriermolecule is phycoerythrin or allophycocyanine. In some embodiments, thecarrier molecule is a quantum dot (Qdot). In some embodiments, thecarrier molecule is a magnetic particle. In some embodiments, thecarrier molecule is a nanoparticle. In some embodiments, the MHCmolecule is an MHC class I molecule. In some embodiments, the MHCmolecule comprises an HLA-A*0201 heavy chain. In some embodiments, theMHC molecule is an MHC class II molecule. In some embodiments, the MHCmolecule is loaded with an antigenic peptide. In some embodiments, theMHC molecule is loaded with a peptide comprising the sequence GILGFVFTLor a peptide comprising a sequence provided in Table 2.

Some aspects of this invention provide a method comprising providing anMHC molecule bound to an antigenic MHC molecule-binding peptide that isconjugated to a tag via a cleavable linker, removing the tag from theantigenic peptide, and conjugating a chelant moiety to a heavy chain ofthe MHC molecule. In some embodiments, the method further comprisescontacting a multivalent chelant molecule with the MHC molecule underconditions suitable for the chelant moiety conjugated to the MHCmolecule to form a chelate complex bind with a chelant moiety of themultivalent chelant molecule.

Some aspects of this invention provide a method comprising contacting apopulation of cells with a protein multimer according to any precedingclaim, wherein the multimer comprises a chelate bond and a detectablelabel, and detecting a cell binding the multimer. In some embodiments,the protein multimer is an MHC multimer. In some embodiments, thedetectable label is a fluorophore. In some embodiments, detecting is byfluorescent microscopy or cell sorting. In some embodiments, thedetectable label is a magnetic particle. In some embodiments, detectingis by isolating the cell binding the multimer. In some embodiments,detecting comprises quantifying a number of cells binding the multimer.In some embodiments, detecting comprises quantifying a number of cellsbinding the multimer as a ratio to a number of cells of the populationof cells that do not bind the multimer. In some embodiments, the methodfurther comprises reversing the binding of the multimer to the cellbinding the multimer by contacting the cells binding the multimer with amonomeric chelant moiety competing for the chelate complex bondcomprised by the multimer. In some embodiments, the monomeric chelantmoiety is an imidazole molecule.

Some aspects of this invention provide a method comprising contacting apopulation of cells with a protein multimer according to any precedingclaim, wherein the multimer comprises a chelate bond and a detectablelabel; optionally, detecting a cell binding the multimer; and isolatingthe cell binding the multimer. In some embodiments, the multimer is anMHC multimer. In some embodiments, the method further comprisesreversing the binding of the multimer to the cell binding the multimerby contacting the isolated cell binding the multimer with a monomericchelant moiety competing for the chelate complex bond comprised by themultimer. In some embodiments, the monomeric chelant moiety is animidazole molecule. In some embodiments, the isolated cell is a T-cell.In some embodiments, the T-cell does not undergo CD8/TCR-mediatedactivation during the contacting, optionally, during the detection, andduring the isolation step. In some embodiments, the isolated T-cell ispart of an isolated, native T-cell population.

Some aspects of this invention provide a method comprising, contacting apopulation of cells expressing a T-cell receptor with an MHC multimerprovided herein under conditions suitable for the multimer to bind tothe T-cell receptor and for a time sufficient for the T-cellreceptor/MHC class I molecule interaction to activate a T-cellexpressing the T-cell receptor and binding the MHC multimer. Someaspects of this invention provide a method comprising, contacting apopulation of cells expressing a T-cell receptor with an MHC multimerprovided herein under conditions suitable for the multimer to bind tothe T-cell receptor and to render the T-cell non-responsive to anaturally occurring antigen. In some embodiments, the method furthercomprises contacting the population of T-cells with an agent able torelease the chelate complex bond of the MHC multimer after the cellswere contacted with the multimer.

Some aspects of this invention provide a cell or cell populationcomprising a cell contacted with the protein multimer of any precedingclaim, wherein the multimer comprises a chelate complex bond. In someembodiments, the multimer is an MHC multimer. In some embodiments, thecell is or has been contacted with an excess of monomeric chelantmoieties competing for the chelate complex bond comprised by themultimer.

Some aspects of this invention provide a kit comprising an MHC multimeras provided in any preceding claim. In some embodiments, the MHCmultimer is loaded with an antigenic peptide. In some embodiments, theMHC multimer is an empty MHC multimer. In some embodiments, the kitfurther comprises at least one antigenic peptide that can be loaded ontothe empty MHC multimer. In some embodiments, the kit comprises 2, 3, 4,5, 6, 7, 8, 9, 10, or more than 10 different antigenic peptides. In someembodiments, the antigenic peptides are antigenic peptides of tumorantigens.

Some aspects of this invention provide an isolated peptide-loaded MHCmolecule comprising an MHC heavy chain, and an antigenic peptide. Insome embodiments, the peptide is conjugated to a tag. In someembodiments, the tag is an affinity tag. In some embodiments, the tag isa desthiobiotin (DTB) tag. In some embodiments, the peptide isconjugated to a tag for ion exchange chromatography. In someembodiments, the MHC molecule is an MHC class II molecule. In someembodiments, the tag is an acidic tag. In some embodiments, the acidictag is an acidic cyanine dye. In some embodiments, the acidic tag is apeptide tag comprising a plurality of acidic amino acid sequences. Insome embodiments, the tag is a pY-D4, pY-D5, pY-D6, pY-D7, pY-D8, pY-D9,or pY-D10 tag. In some embodiments, the tag is a pY-E4, pY-E5, pY-E6,pY-E7, pY-E8, pY-E9, or pY-E10 tag. In some embodiments, the MHCmolecule further comprises a heavy chain that is conjugated to a chelantmoiety. In some embodiments, the molecule comprises a combination of aheavy chain and an antigenic peptide discloses in Table 2. In someembodiments, the tag is conjugated to the peptide via a cleavablelinker. In some embodiments, the linker is a photocleavable linker. Insome embodiments, the linker is an NP A linker. In some embodiments, thelinker is a peptide linker that comprises an amino acid sequence thatcan be cleaved by a protease or by a chemical. In some embodiments, theMHC molecule is comprised in an MHC multimer.

Some aspects of this invention provide a method comprising contacting anempty MHC molecule with an antigenic peptide conjugated to a tag underconditions suitable for the antigenic peptide to bind the MHC molecule.In some embodiments, the MHC molecule is an MHC class II molecule. Insome embodiments, the tag conjugated to the MHC class II bindingantigenic peptide is an affinity tag that is not a polyhistidine tag. Insome embodiments, the tag is a desthiobiotin (DTB) tag. In someembodiments, the tag is an acidic tag. In some embodiments, the acidictag is an acidic cyanine dye. In some embodiments, the acidic tag is apeptide tag comprising a plurality of acidic amino acid sequences. Insome embodiments, the tag is a pY-D4, pY-D5, pY-D6, pY-D7, pY-D8, pY-D9,or pY-D10 tag. In some embodiments, the tag is a pY-E4, pY-E5, pY-E6,pY-E7, pY-E8, pY-E9, or pY-E10 tag. In some embodiments, the tag isconjugated to the peptide via a cleavable linker. In some embodiments,the linker is a photocleavable linker. In some embodiments, the linkeris an NP A linker. In some embodiments, the linker is a peptide linkerthat comprises an amino acid sequence that can be cleaved by a proteaseor by a chemical. In some embodiments, the tag is a part of a cleavablelinker that remains after cleavage of the linker.

Some aspects of this invention provide a method comprising contacting anMHC class II type alpha heavy chain with an MHC class II type beta heavychain under conditions suitable for the alpha and the beta chain to forma heterodimeric MHC class II molecule. In some embodiments, at least oneof the MHC class II heavy chains is conjugated to a tag, and isolatingthe MHC class II molecule, wherein the isolating comprises a step ofaffinity chromatography. In some embodiments, the tag is a protein orpeptide tag. In some embodiments, the tag is a poly-His tag. In someembodiments, the His tag comprises 3-12 His residues. In someembodiments, the affinity chromatography is Ni2+-NTA chromatography. Insome embodiments, the contacting is performed by expressing both heavychains in a cell. In some embodiments, the cell is an insect cell. Insome embodiments, the MHC class II molecule is “empty” (not loaded withan antigenic, MHC class II binding peptide). In some embodiments, themethod further comprises contacting the MHC class II molecule with anMHC class II binding antigenic peptide. In some embodiments, the MHCclass II binding antigenic peptide is conjugated to a tag. In someembodiments, the tag is an affinity tag. In some embodiments, the tagconjugated to the MHC class II binding antigenic peptide is an affinitytag that is not a polyhistidine tag. In some embodiments, the tag is adesthiobiotin tag. In some embodiments, the tag is an acidic tag. Insome embodiments, the acidic tag is an acidic cyanine dye. In someembodiments, the acidic tag is a peptide tag comprising a plurality ofacidic amino acid sequences. In some embodiments, the tag is a pY-D4,pY-D5, pY-D6, pY-D7, pY-D8, pY-D9, or pY-D10 tag. In some embodiments,the tag is a pY-E4, pY-E5, pY-E6, pY-E7, pY-E8, pY-E9, or pY-E10 tag. Insome embodiments, the tag is conjugated to the peptide via a cleavablelinker. In some embodiments, the tag is a desthiobiotin tag.the linkeris a photocleavable linker. In some embodiments, the tag is adesthiobiotin tag.the linker is an NPβA linker. In some embodiments, thetag is a desthiobiotin tag.the linker is a peptide linker that comprisesan amino acid sequence that can be cleaved by a protease or by achemical. In some embodiments, the tag is a desthiobiotin tag.the tag isa part of a cleavable linker that remains after cleavage of the linker.

The subject matter of this application may involve, in some cases,interrelated products, alternative solutions to a particular problem,and/or a plurality of different uses of a single system or article.Other advantages, features, and uses of the invention will becomeapparent from the following detailed description of non-limitingembodiments of the invention when considered in conjunction with theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. MHC-peptide complexes under study. A) Structure of aNTA-Ni²⁺-His₆ complex in which Ni²⁺ forms a chelate on one hand with thethree carboxyl moieties of NTA and with the imidazole groups of twohistidines on the other. B) The conventional biotin-streptavidin PE-MHCclass I-peptide multimer. C) NTA multimers in which His taggedMHC-peptide monomers are bound to NTA-PE, obtained by reacting PEstreptavidin with biotin-NTA compounds. D) As C), but the NTA moietiesare directly conjugated to PE. E) As D) using Qdots instead of PE.

FIG. 2. His tagged HLA-A2 under study and their refolding efficiencies.A) Composition of the different C-terminal tags on HLA-A2 including thebiotinylation sequence peptide BSP, linear His₆, His₁₂ or two His6 tagsseparated by a flexible GGGSGGGSGS (SEQ ID NO:4) spacer. The underlinedresidues (274-276) mark the C-terminus of the A2 α3 domain. Sequences,from top to bottom, correspond to SEQ ID NOs 5-8. B) HLA-A2 heavy chainscontaining the different tags were refolded with β2m in the presence ofFlu matrix₅₈₋₆₆ peptide. The refolding efficiencies expressed as percentwith 100% being the one of HLA-A2-BSP.

FIG. 3. Structures of the NTA linker under studies. A) Biotin-mono NTAin which a biotin (circled) is coupled to NTA-lysine viaN-(5-(3-maleimidopropionylamino)-1-carboxy-pentyl)iminodiacetyl-cysteine-Nα-acetyl-Nε-amino-caproyl.B) biotin-di-NTA in which two NTA lysines were coupled via maleimide totwo cysteines spaced by a glycine. C) biotin-tetra-NTA in which twodi-NTA moieties are joined via GGGSGGGSGS (SEQ ID NO: 9) spacer. D)Amino-di-NTA, analogous to B) with a free amino group (circled). E)thiol-di-NTA, analogous to D) but with an N-terminal cysteine containinga free thiol (circled). F) Commercially available NTA-biotin (BiotiumInc, Hayward, Calif.).

FIG. 4. SPR binding studies for the different Ni-NTA linkers and Histags. A) Experimental set up consisting in first loading streptavidincoated sensor chips with biotin-mono-NTA or biotin-di-NTA, followed bysaturating with NiCl₂ and washing. The changes in resonance units (RU)were measured upon injecting the different A2/Flu complexes over theNTA-Ni²⁺ loaded chips. B) Compilation of the dissociation constants(K_(D) in nM), binding on-rate constants (k_(on) in M⁻¹×sec⁻¹×10⁴) andbinding off-rates (k_(off) in sec⁻¹×10⁻³) recorded at room temperaturefor the differently His tagged A2/Flu complexes on mono-NTA (FIG. 3A) ordi-NTA (FIG. 3B) loaded sensor chips.

FIG. 5. Bindings isotherms of different NTA-biotin-streptavidin PEA2/Flu₅₈₋₆₆ multimers. A) Cloned, Flu-specific 81P1 cells were incubatedat 20° C. for 30 min with graded concentrations of PE streptavidinA2/Flu₅₈₋₆₆ multimers containing NTA₂×2×His₆ (dark ▴), NTA₂×His₁₂ (light▪), NTA×2×His₆ (light ▴); NTA×His₁₂ (dark ▪), NTA₂×His₆ (light ♦) orNTA×His₆ (dark ♦). For comparison BSP multimers were included (circles).After washing, cell-associated fluorescence was assessed by flowcytometry. B) Alternatively 20° C. binding isotherms were assessedlikewise on cloned BCB 70 cells for multimers containing NTA₂-biotin(light lines) or NTA₄-biotin (dark lines) and A2/Flu₅₈₋₆₆ complexes withHis₁₂ (♦) or 2×His₆ tags (▴).

FIG. 6. Dissociation kinetics of different NTA₂-biotin-steptavidinA2/Flu₅₈₋₆₆ multimers. A, B) Cloned Flu-specific 81P1 cells wereincubated at 4° C. for 1 h with multimers (5 nM) containing biotin NTA₂and His₁₂ (A) or 2×His₆ (B) tagged A2/Flu complexes. After washing thecells were incubated at 4° C. in HBSS in the absence (squares) orpresence of 50 mM imidazole (triangles), 50 mM imidazole +20 mM EDTA(dashes) or 100 mM imidazole (diamonds) and after the indicated periodsof time cell-associated multimers were assessed by flow cytometry. Forcomparison conventional BSP multimers were included (circles). Theinserted numbers indicate the times (t_(1/2)) at which half maximaldissociation was observed.

FIG. 7. Dissociation kinetics of NTA₂- and NTA₄ biotin-streptavidinA2/Flu₅₈₋₆₆ multimers. A, B) Cloned BCB 70 cells were incubated at 4° C.for 1 h with NTA₂-biotin-streptavidin PE (A) or NTA₄-PE A2/Flu multimers(B) containing the His₁₂ (dark symbols) or 2×His₆ tag (light symbols).After washing the cells were incubated at 4° C. in HBSS in the absence(diamonds) or presence of 50 mM imidazole (triangles), or 100 mMimidazole (squares) and after the indicated periods of timecell-associated multimers were assessed by flow cytometry. Forcomparison conventional BSP multimers were included (circles). Theinserted numbers indicate the times (t_(1/2)) at which half maximaldissociation was observed.

FIG. 8. Preparation of NTA₂-PE conjugates. A) Structure of PE, afluorescent protein of 240,000 Da, has a rigid structure and 24 surfaceexposed lysines. B) Strategy to couple NTA₂-cysteine (FIG. 3E) on PE. Ina first step PE was reacted with the maleimide N-hydroxy-succinimidylester SM(PEG)₂ (succinimidyl-maleido(PEG)₂), whereby maleimide groupsare conjugated onto PE lysines. In a second step the NTA₂ cysteine (FIG.3E) was added, which by reacting with maleimides on PE forms stablethio-ethers.

FIG. 9. Binding isotherms of NTA₂-PE A2/Flu₅₈₋₆₆ multimers. Cloned 81P1cells were incubated for 45 min at the indicated temperatures withgraded concentrations of conventional BSP multimers (circles), or withmultimers containing NTA₂-PE coupled PE and A2/Flu complexes containingthe 2×His₆ tag (squares) or the His₁₂ tag (diamonds). After washing thecells they were analyzed by flow cytometry.

FIG. 10. Dissociation kinetics of NTA₂-PE A2/Flu₅₈₋₆₆ multimers. ClonedBCB 70 cells were incubated for 1 h at 4° C. with conventional BSPmultimers (circles) or multimers containing NTA₂-PE conjugated withA2/Flu complexes containing a 2×His₆ tag. After washing the cells theywere incubated at the indicated temperatures in FACS buffer containingor not (squares) 50 mM imidazol (triangles) or 100 mM imidazol(diamonds). After the indicated periods of time the cells were analyzedby flow cytometry.

FIG. 11. FACS sorting with reversible NTA₂-PE but not conventionalA2/Flu multimers provides high cell viability. A) Cloned BCB 70 cellswere FACS sorted upon staining with the conventional A2/Flu₅₈₋₆₆ BSP(black bars) or NTA₂-PE-2×His₆ multimers (dark gray bars), after 1 washwith 100 mM imidazol the cells were cultured and at the indicatedintervals viable cells enumerated by trypan blue exclusion counting. Ascontrols cells were left untreated (white bars) or washed 1× with 100 mMimidazol (light gray bars).

FIG. 12. Synthesis of mono-NTA and di-NTA-biotin. The Compounds shown inFIGS. 3A and B were synthesized using: i) solid phase peptide synthesison chloro-trityl resin; ii) Fmoc for transient protection and ii)coupling with TBTU (2 eq), HOBt (2 eq), DIEA (4 eq) (a) and fordeprotection TFA/H₂O/TIPS (92.5/2.5/5) (b). The intermediary thiolcompounds were reacted with maleimido-NTA-lysine in 100 mM phosphatebuffer, pH 7.2 (c). The distance x is about 16 {acute over (Å)} and y 23{acute over (Å)}.

FIG. 13. Synthesis of an alternative di-NTA-biotin. An alternativedi-NTA-biotin was synthesized starting on a rink amide chloro-tritylresin to prepare the peptide back bone (a), which was reacted insolution with tri-tert. butyl-NTA lysine (b), followed by completedeprotection (c); in this compound the distance x′ is about 19 {acuteover (Å)} and y′ 9 {acute over (Å)}.

FIG. 14. Synthesis of NTA₄-biotin. In a first step backbone peptide wassynthesized by solid phase peptide synthesis on a chloro-trityl resin (). The deprotected and HPLC purified peptide was reacted in phosphatebuffer with 8 equivalents of NTA maleimide (b). The molecules comprisestwo di-NTA moieties joined by a flexible GGSGGGSGS (SEQ ID NO: 10)linker, which is the same as in the 2×His6 tag (FIG. 2A).

FIG. 15. Multimers containing the short di-NTA linker more avidly staincloned CD8+ T cells than those containing the long di-NTA linker A, B)The experiment shown in FIG. 5A was repeated using multimers containingeither the short di-NTA-biotin (A) (FIG. 3B) or the long one (B) (Suppl.Fig. S2) and A2/Flu₅₈₋₆₆ monomers carrying the 2×His₆ tag (triangles),His₁₂ tag (squares) or a His6 tag (diamonds). For comparisonconventional BSP multimers were included (red circles).

FIG. 16. BCB 70 CTL were incubated likewise with streptavidin coatedQdots₆₀₅ were loaded with NTA₂-biotin and conjugated with A2/Flu₅₈₋₆₆monomers containing the indicated tags; after washing the cells wereanalyzed by flow cytometry. For comparison Qdots loaded withbiotinylated monomers were included (BSP).

FIG. 17. Impact of the degree of NTA₂ conjugation of PE on NTA₂-PE Histag multimer staining. PE was conjugated with di-NTA as shown in FIG. 8using a constant concentration of PE (10 nM) and the indicatedconcentrations of SM(PEG)₂ for 2 h at ambient temperature, followed byalkylation NTA₂-cysteine. The resulting NTA₂-PE conjugates were loadedwith A2/Flu₅₈₋₆₆ monomers containing the indicated His tags, themultimers (5 nM) were incubated at 20° C. for 30 min with cloned 81P1cells. After washing cell-associated multimers were measured by flowcytometry. For comparison conventional BSP multimers were included.

FIG. 18. Binding isotherms of different A2/Flu multimers on a polyclonalFlu-specific CD8+ T cell population. A polyclonal population ofFlu-specific CD8+ T cells was incubated at 20° C. for 30 min with A2/Flumultimers containing NTA₄-biotin and 2×His₆ tag (light triangles,NTA₄-biotin and His₁₂ (light diamonds), NTA₂-biotin and 2×His₆ tag (darktriangles), NTA₂-biotin and His₁₂ tag (dark diamonds), NTA2-PE and2×His₆ tag (squares) and conventional BSP multimers (circles). Cellassociated fluorescence was measured by flow cytometry.

FIG. 19. Dissociation kinetics of A2/Flu₅₈₋₆₆ DTB and NTA₂-PE multimers.A, B) Cloned BCB 70 cells were incubated at 4° C. for 1 h with 10 nM ofA2/Flu₅₈₋₆₆ multimers containing DTB streptavidin PE (A) or NTA₂-PE and2×His₆ (B). After washing the cells were incubated at 4° C. (diamonds),20° C. (squares) or 37° C. (triangles) in FACS buffer supplemented with2 nM biotin (in A) or 100 mM imidazol (in B). Cell associatedfluorescence was determined by flow cytometry after the indicatedperiods of incubation.

FIG. 20. Preparation of immunopure biotin-streptavidin multimers. Emptysoluble MHC II molecules containing C-terminal leucine zippers arepurified from supernatants by immuno-affinity chromatography and loadedwith a peptide conjugated with His₆ tag via a photo-cleavable linker.The complexes containing this peptide are isolated by affinitychromatography on a Ni²⁺-NTA, thereafter the tag is removed byphotolysis. The resulting immunopure MHC II peptide monomers areenzymatically biotinylated and multimerized with PE streptavidin.

FIG. 21. Photochemical removal of tags from MHC II binding peptides. A)To allow photochemical removal of tags from MHC II-restricted peptides,these were added via 2-nitro-phenyl-β-Ala (NPβA); upon UV irradiationthis residue is cleft, such that the N-terminal fragment is an amid andthe C-terminal one carries a 2-nitroso-phenacetyl-β-acetoyl group. B)HLA-DR4 loaded with H₆-GSG-NPβA-HA₃₀₆₋₃₁₈peptide was irradiated at365+/−40 nm for the indicated periods of time and the % of complexescarrying the His tag was assessed by ELISA using a His tag-specific mAb.C) the same experiment was performed for DR4-HA₃₀₆₋₃₁₈-NPβA-GSG-H₆complexes. The inserted numbers (t_(1/2)) indicate the time at whichhalf maximal photolysis occurred.

FIG. 22. Comparative staining of HA clones by conventional andimmunopure DR4-HA multimers. A-D) The indicated HA₃₀₆₋₃₁₈-specificDR4-restricted Th1 clones were incubated at 37° C. for 2 h with theindicated concentrations of the DR4 multimers containing: HA(conventional) (solid circles), H₆-GSG-NPβA-HA₃₀₆₋₃₁₈(immunopure)(triangles), H₆-GSG-NPβA-HA₃₀₆₋₃₁₈ (immunopure; after UV irradiation)(inverted triangles), HA₃₀₆₋₃₁₈-NPβA-GSG-H₆(immunopure) (diamonds),HA₃₀₆₋₃₁₈-NPβA-GSG-H₆ (immunopure, after UV irradiation) (two-coloredcircles). The cells were washed and the cell bound multimers (MFI)assessed by flow cytometry. E) The TCR and CD4 expression of the cloneswere assessed by flow cytometry and the half maximal IFNγ response(EC50) by ELISA.

FIG. 23. Preparation of immunopure DR4-HA306-318 monomers using Cy5.5.tagged peptides. A) Empty MHC II molecules are loaded with Cy5.5 taggedpeptide and subjected to GFC and anion exchange chromatography. Cy5.5contains four negative charges, i.e. is strongly negatively charged. B)GFC on a Superdex S75 column of DR4 after loading withHA₃₀₆₋₃₁₈-GSGC-Cy5.5 recording of the OD280 (black, protein) and OD675nm (gray, Cy5.5). C) Assessment of MHC II protein (black bars) and Cy5.5(gray bars) by ELISA using mAb specific for DR4 and Cy5.5, respectivelyof HA₃₀₆₋₃₁₈-GSGC-Cy5.5, DR4-HA; DR52b-ESO₁₂₃₋₁₃₇-GSGC-Cy5.5 andDR52b-ESO₁₂₃₋₁₃₇. D) Anion exchange chromatography on a Mono-Q columnusing the indicated NaCl gradient and recording the OD280 (protein) andOD675 nm (Cy5.5) of the eluant.

FIG. 24. Preparation of immunopure MHC II-peptide NTA multimers usingthe pY-D4-tag. Empty MHC II molecules containing a C-terminal His tag atthe acidic leucine zipper are purified by affinity chromatography on NTAcolumns and loaded with given peptides (e.g. HA₃₀₆₋₃₁₈) containing thestrongly negatively charged phospho-tyrosine-Asp₄ (pY-D₄) tag. Thepeptide loading is monitored by ELISA using anti-pY mAb. Immunopuremonomers are obtained by anion exchange chromatography, from which thepY-D₄ tag is removed by UV irradiation and NTA multimers upon reactionwith NTA-PE or NTA Qdots.

FIG. 25. Preparation and testing of immunopure DR4/HA₃₀₆₋₃₁₈ NTAmultimers. A) empty, His tagged DR4 molecules were purified on aNi²⁺-NTA affinity column, which was eluted with 200 mM imidazol,monitoring the OD280 nm of the eluate. B) The eluted DR4 was subjectedto GFC on a Superdex S200 column again monitoring the OD280 nm of theeluate. C) The collected fractions (gray lines) were analyzed bySDS-PAGE (10% non-reducing) applying a low (2 μg/ml) or high (5 μg/ml)concentration. D) Empty DR4 was loaded with pY-D₄-GSG-NPβA-HA₃₀₆₋₃₁₈peptide followed by purification by anion exchange chromatography onMono-Q column, which was eluted with the indicated NaCl gradient (upperpanel). Fractions (0.5 ml) were collected and their content of DR4(middle panel) and pY (phospho-tyrosine) (lower panel), respectively,determined by ELSA as shown in the lower two panels.

FIG. 26. Impact of peptide loading on tetramer purity. The purity oftetramers decreases rapidly with decreasing peptide loading.

FIG. 27. Effect of His tags on peptide binding to DR1, DR4 and DR52b.The binding of the indicated NY-ESO-1 peptides carrying a N-terminalHis₆ tag or not to DR1, DR4 and DR52b was determined in a competition asdescribed in Materials and Methods.

FIG. 28. Neuraminidase treatment increases DR4/HA₃₀₆₋₃₁₈ multimerbinding. A) The indicated DR4-restricted, HA-specific T cell clones werepretreated or not with neuraminidase (30 min incubation at 37° C. with0.03 U/ml of neuraminidase) and incubated with conventionalDR4/HA₃₀₆₋₃₁₈ multimers (20 μg/ml) at 37° C. for 2 h. After washingcell-associated multimers (MFI) were assessed by flow cytometry. B,C)Cloned 10.5 (A) or 7.1 cells were incubated likewise with the indicatedconcentrations of conventional DR4/HA₃₀₆₋₃₁₈ multimers and cellassociated fluorescence was assessed by flow cytometry.

FIG. 29. Staining is a function of PE substitution degree.

FIG. 30. Staining isotherms on Flu stimulated PBMC with differentmultimers at different concentrations. The x-axis provides the multimerconcentration (in nM).

FIG. 31. Exemplary NTA linkers.

FIG. 32. Staining performance of PE conjugates comprising A2/Flu₅₈₋₆₆monomers, or DR4/Flu HA₃₀₆₋₃₁₈ monomers.

FIG. 33. Comparison of conventional, PE-Cys-PEG2-NTA2 and PE-HNO-NTA2multimers staining.

FIG. 34. Reduction of background staining by milk supplements.

FIG. 35. Binding titration of NTAmers to determine best dilution for exvivo staining.

FIG. 36. Ex vivo staining with BSP and NTA multimers on fresh peripheralblood mononuclear cells (PBMCs).

FIG. 37. Overview of an exemplary strategy for NTA-His tag multimerpreparation using desthiobiotin (DTB).

FIG. 38. Structures of biotin and DTB.

FIG. 39. Evaluation of DTB tag peptide purification efficiency.

FIG. 40. Schematic of 2 ml of StreptActin High Capacity sepharose (IBA)

FIG. 41. Quantification of flowthrough, washing and elution of aDTB-tagged peptide.

FIG. 42. Purification of MHC I-DTB peptide complexes on StreptActinsepharose.

FIG. 43. Exemplary scheme of DTB-HA peptide loading and purification.

FIG. 44. Generation and purification of DR4-DTB-HA peptide complexes.

FIG. 45. NTA moieties used in PE-NTA₂ and biotin-NTA₄-SA-PE multimers.

FIG. 46. Staining of Flu HA₃₀₆₋₃₁₈-specific CD4⁺ T cells withDR4/HA₃₀₆₋₃₁₈ BSP, biotin-NTA₄-SA-PA, or PE-NTA₂ multimers made withDTB-streptactin purified monomers.

FIG. 47. Staining of HA-specific CD4⁺ T cell clones 9(A) or 8(B) withdifferent concentrations of DR4/HA₃₀₆₋₃₁₈ BSP, biotin-NTA₄-SA-PA, orPE-NTA₂ multimers. Scatchard analysis was performed and the K_(D)(dissociation constant) and B_(max) (maximal binding) values aredescribed in the tables on the right.

FIG. 48. Background staining of DR4/HA₃₀₆₋₃₁₈ SA-PE NTA₄ and PE NTAmultimers. Background staining was efficiently suppressed by addition of0.5% milk powder (see FIG. 34).

FIG. 50. Reversibility of multimer staining. Biotin-NTA₄, but not BSPmultimers, can be rapidly removed from stained, antigen-specific cells.

FIG. 51. Comparative staining of a DR1/ESO₁₁₉₋₁₄₃ cell line byDR1/NY-ESO₁₁₉₋₁₄₃ BPS, PE-NTA₂ and biotin-NTA₄-SA-PE multimers.

FIG. 52. Evaluation of additional tags for purification ofpeptide-loaded MHC monomers.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Some aspects of this invention relate to protein multimers in whichconjugation of a plurality of monomeric proteins is based on chelatecomplex bonds between the monomeric proteins and a central carriermolecule. Methods for generation and the use of such protein multimersare also provided herein.

Some aspects of this invention provide “reversible” protein multimersthat can be dissociated by releasing the chelate complex bonds betweenthe carrier molecule and the monomeric proteins, for example, bywithdrawal of a central ion from the chelate complex bond or bycontacting the chelate complex with a free chelant that can displace oneof the binding partners forming the chelate complex. This chelate bondrelease results in re-monomerization of the proteins comprised in themultimer. In some embodiments, multimer assembly and/orre-monomerization are carried out under non-denaturing, physiological,and/or non-toxic conditions, rendering the respective multimers suitablefor in vivo, ex vivo, and in vitro applications involving living cellsand/or monomeric proteins prone to denaturation.

Some aspects of the invention provide, for the first time, thatreversible MHC-peptide multimers can be built on Ni²⁺ NTA-His tagcomplexes that exhibit equal or superior staining properties as comparedto conventional multimers. These novel staining reagents are fullyreversible in that they can be rapidly dissociated into monomericsubunits upon addition of imidazol, which allows, for example, sortingof bona fide antigen-specific CD8+ T cells.

Some aspects of this invention provide methods and materials for thepreparation of fluorescent MHC protein multimers, in which conjugationis based on chelate complex formation between nitrilotriacetic acid(NTA) and an amino acid sequence comprising a polyhistidine sequence,also referred to as a histidine tag (His tag). In some embodiments, thechelate complexes are formed in the presence of a Ni²⁺ cation. In someembodiments, the His tag comprises 3-12 His residues. In someembodiments, the His tag is a hexahistidine (His₆) tag or a 2×His₆ tag,comprising to hexahistidine sequences separated by a short amino acidlinker.

Some aspects of this invention are based on the recognition thatreversible protein multimers based on chelate complex bonds are stableenough to be useful for various applications, for example, cell orprotein staining and isolation procedures, but can be dissociated underphysiological, non-toxic conditions, for example, by withdrawing acentral ion that is required for the formation of the chelate complex orby contacting them with an agent competing for the chelate complex bond,and thus releasing the bond between carrier molecule and monomericprotein. For example, the reversible Ni²⁺ NTA-His tag interaction-basedmultimers described herein are “reversible” in that they can bedissociated either by withdrawing the chelant cation (e.g. Ni²⁺ or Co²⁺)or by adding free competing chelant, for example, free imidazol, whichdisplaces the His tag from the chelate complex. Imidazol is commerciallyavailable and is commonly used for purification of recombinant proteinson Ni²⁺ NTA affinity chromatography (see e.g.products.invitrogen.com/ivgn/product/K95001?ICID==Search-Product; andQIAExpressionist Handbook, Qiagen Inc., March 2001, available fromwww.Qiagen.com; both incorporated herein in their entirety byreference).

For example, the interaction of one Ni²⁺ NTA with a hexahistidine tag(His₆) has a dissociation constant (K_(D)) of about 10⁻⁶-10⁻⁷ M and issufficiently stable to allow purification of His tagged recombinantproteins from culture supernatants (9, 10). Previous studies haveinvestigated ways to render this interaction more stable. For example,different His tags have been examined and it has been shown thatincreasing the length of the His tag increases the stability of thecomplexes (9). In particular it has been demonstrated that linking twoHis₆ tags via a flexible linker, such as GGGSGGGSGS (SEQ ID NO: 11)provides a high increase in stability (11, 12). Further, linkers havebeen synthesized that contain two to four NTA groups (13-15). Thebinding to His tags, in particular longer ones, increases considerablywith the number on Ni²⁺ NTA entities. While, in some embodiments, theHis tags are expressed tethered, or fused to a recombinant protein, theNTA compounds have to be synthesized, as only mono-NTA (NTA1)derivatives are commercially available. Some aspects of this inventionprovide optimized configurations of NTA bound to a linker and optimizedpairings of specific NTA configurations with specific His-tags toachieve a modulation of chelate complex bond stability over multipleorders of magnitude. Accordingly, reversible multimers, as providedherein, can be customized to fulfill specific requirements of a widevariety of research, diagnostic, and therapeutic applications.

In some embodiments, reversible MHC multimers, for example, reversibleMHC class I multimers, are provided. In some embodiments,fluorescently-labeled soluble MHC peptide multimers are provided. Insome embodiments, methods for the use of reversible MHC multimers areprovided, for example, methods useful to quantitate, isolate and/orcharacterize antigen-specific T-cells, for example, CD8⁺ and CD4⁺ Tcells. In some embodiments, methods for the use of MHC multimers areprovided that are useful for phenotypic T-cell analysis or for theanalysis of T-cell receptor (“TCR”) repertoire in a subject.

Conventional MHC multimers are typically prepared by enzymaticbiotinylation of monomeric MHC proteins comprising a C-terminalbiotinylation sequence peptide (BSP) and subsequent conjugation withstreptavidin bound to a fluorescent dye, typically phycoerythrin (PE) orallophycocyanine (APC). Due to the large size of PE and APC, conjugateswith streptavidin vary in stoichiometry, accessibility, and orientationof the biotin binding sites, often resulting in variable valency andinhomogeneous populations of MHC multimers. Other types of PE- orAPC-based MHC-peptide staining reagents have been described, includingStreptamers, desthiobiotin (DTB) multimers, pentamers (Proimmune Inc.,FL, USA). Non-phycobilin-based MHC multimers have also been developed,for example Quantum dots loaded with MHC class I-peptide complexes,which allow simultaneous use of multiple MHC class I-peptidespecificities in polychrome flow cytometry, Cy5-labeled dimeric,tetrameric and octameric MHC class I-peptide complexes, dextramers(Immudex, Copenhagen, Denmark) and dimeric MHC-peptide-immunoglobulin(Ig) fusion proteins.

While exemplary reversible multimers described herein include reversibleMHC protein multimers, it will be apparent to those of skill in the artthat the methods and reagents provided herein can be applied to generatereversible multimers of proteins other than MHC proteins. The methodsfor the generation and use of protein multimers, accordingly, areuniversally applicable to proteins of different nature, for example, tobinding proteins, such as MHC proteins, antibodies, antibody fragments,ligands, adnectins, and receptors or receptor fragments. Exemplarymultimers of such binding proteins, namely of peptide-loaded or emptyMHC molecules, are provided in the working example section. Further,those of skill in the art will appreciate that virtually any kind ofprotein can be engineered to form a reversible multimer as provided byaspects of this invention as long as the monomeric protein is amenableto conjugation to a chelant moiety, for example, to addition of aHis-tag fusion.

Accordingly, those of skill in the art will appreciate that the methodsfor the generation of protein multimers described herein can be appliedto the generation of additional chelate complex bond-based bindingmolecules. For example, some embodiments, provide multimers of proteinor non-protein binding molecules. The term “binding molecule” as usedherein, refers to a molecule that is able to bind a binding partner vianon-covalent interaction. Typically, in the context of cells, cellculture, or processing of living cells (e.g. staining, FACS sorting), abinding molecule is able to form a binding interaction with a bindingpartner that is strong enough to be stable under physiologicalconditions or under the conditions typically encountered during cellprocessing. In some embodiments, a binding molecule binds its bindingpartner with high specificity and/or high affinity. Non-limitingexamples of binding molecules are antibodies and antibody fragments(e.g., Fab, F(ab)′2, single chain antibodies, diabodies, etc.),receptors, proteins binding a ligand, aptamers, and adnectins. The term“ligand” is art-recognized and refers to a binding partner of a bindingmolecule. Ligands can be, for example, proteins, peptides, nucleicacids, small molecules, and carbohydrates. Avidins, for example,streptavidin, are non-limiting examples of binding molecules that canbind a ligand, in this case, for example, biotin.

For example, in some embodiments, reversible antibody fragment, forexample, Fab fragment, multimers are provided, in which a plurality ofFab proteins is bound to a central carrier molecule via chalet complexbonds, for example, NTA-His bonds. In some embodiments, the carriermolecule is a fluorescent microsphere, for example, a FluoSpheres® typefluorosphere (seehttp://products.invitrogen.com/ivgn/product/F8781?ICID=search-product).

Protein Multimers

Some aspects of this invention provide reversible protein multimers inwhich a plurality of proteins is conjugated to a central carriermolecule via a non-covalent binding interaction that can be releasedunder physiological conditions, for example, by contacting the multimerwith an agent able to displace one of the binding partners from thebinding interaction.

The terms “protein,” “polypeptide,” or “peptide,” as used herein, referto a polymer of at least three amino acid residues linked together bypeptide bonds. The terms are interchangeably used herein and refer toproteins, polypeptides, and peptides of any size, structure, orfunction. Typically, a protein will be at least three amino acids long.In some embodiments, inventive proteins contain only natural aminoacids, although in other embodiments non-natural amino acids (e.g.,compounds that do not occur in nature but that can be incorporated intoa polypeptide chain; see, for example, U.S. Pat. No. 7,045,337, whichdescribes incorporation of non-natural amino acids into proteins) and/oramino acid analogs as are known in the art may alternatively beemployed. In some embodiments, one or more of the amino acids in aninventive protein are modified, for example, by the addition of achemical entity such as a carbohydrate group, a hydroxyl group, aphosphate group, a farnesyl group, an isofarnesyl group, a fatty acidgroup, a linker for conjugation, functionalization, or othermodification, etc. A protein may also be a single molecule or may be amulti-molecular complex. A protein may be just a fragment of a naturallyoccurring protein or peptide. A protein may be naturally occurring,recombinant, or synthetic, or any combination of these. In someembodiments, the protein is an MHC molecule.

The term “conjugated,” as used herein, refers to an entity, molecule, ormoiety that is stably associated with another molecule or moiety via acovalent or non-covalent bond. In some embodiments, the conjugation isvia a covalent bond, for example, in the case of a peptide tagconjugated to an MHC protein via fusion of the peptide to a heavy chainof the MHC protein. In other embodiments, the conjugation is via anon-covalent interactions, for example, via hydrogen bonding, van derWaals interactions, hydrophobic interactions, magnetic interactions, orelectrostatic interactions.

The term “carrier molecule,” as used herein in the context of multimers,refers to a molecule that binds or is conjugated to a plurality ofmonomeric molecules or entities, or a plurality of binding molecules ormoieties that can bind such monomeric molecules or entities. In someembodiments, the carrier molecule is a monomeric molecule, for example,a single molecule of a fluorescent dye. In other embodiments, thecarrier molecule is a multimeric molecule, for example, a polymer, or ananocrystal. For example, in some embodiments, the carrier molecule is afluorophore, for example, a phycobilin, conjugated to a plurality ofbinding molecules, for example, streptavidin. The streptavidinmolecules, in turn, can bind monomeric molecules, for example,biotin-conjugated MHC monomers. In some embodiments, the carriermolecule is a multivalent chelant molecule. In some embodiments, thecarrier molecule is a fluorescent microsphere, for example, aFluoSpheres® type fluorosphere (seehttp://products.invitrogen.com/ivgn/product/F8781?ICID=search-product).In some embodiments, the carrier molecule, for example, a fluorophore,is conjugated to a plurality of chelant molecules or moieties, forexample, NTA molecules that can form chelate complex bonds withhistidine residues in the presence of a divalent cation.

Some aspect of this invention provide optimized chelate complex bondformation between NTA and His chelants by using certain chelantconfigurations. The term “chelant configuration,” as used herein refersto the number and spacing of chelant molecules or moieties in a givenstructure. For example, if chelant moieties, for example, NTA moieties,are conjugated to a carrier molecule, for example, a multivalent bindingmolecule or a fluorophore, via a linker, then a configuration in which asingle chelant moiety is conjugated to the carrier molecule via a singlelinker is referred to as mono-configuration (e.g. mono-NTA, or NTA₁), aconfiguration in which two chelant moieties are conjugated to thecarrier molecule via a single linker is referred to as di-configuration(e.g. di-NTA, or NTA₂), a configuration in which four chelant moietiesare conjugated to the carrier molecule via a single linker is referredto as tetra-configuration (e.g. tetra-NTA, or NTA₄), and so forth.According to some aspects of this invention, different NTA chelantconfigurations and linker structures affect chelate complex bondproperties, including bond strength and, thus, bond stability andreversibility. Similarly, the number and configuration of Histidineresidues in a His tag has been described to affect chelate complex bondproperties. In some embodiments, the His tag is a His6 tag, comprising 6contiguous His residues, a His12 tag, comprising 12 contiguous Hisresidues, or a 2×His6 tag, comprising two sequences of 6 contiguous Hisresidues linked by a short spacer sequence as described in more detailelsewhere herein.

The term “divalent cation” as used herein, refers to an ion that lackstwo electrons as compared to the neutral atom. Examples of divalentcations useful in some embodiments of this invention are Ni2+, Cu2+,Zn2+, Co2+, Cd2+, Sr2+, Mn2+, Fe2+, Mg2+, Ca2+, and Ba2+. Other usefuldivalent cations will be apparent to those of skill in the art and theinvention is not limited in this respect.

In some embodiments, the carrier molecule is a monomeric carriermolecule. In some embodiments, the carrier molecule is a multimeric orpolymeric carrier molecule. For example, in some embodiments, thecarrier molecule is a tetrameric or a hexameric molecule, for example, afluorophore. In some embodiments, the carrier molecule is a fluorophore,a phycobilin, phycoerythrin or allophycocyanine, a nanocrystal, aquantum dot (Qdot), a magnetic particle, or a nanoparticle. The terms“quantum dot” and “Qdot,” as used herein, refer to fluorescent inorganicsemiconductor nanocrystals in which the excitons are confined in allthree spatial dimensions and which are useful as detectable agents insome embodiments of the invention. In some embodiments, the Qdotcomprises CdSe or CdTe. In some embodiments, the Qdot comprises InP orInGaP. In some embodiments, the Qdot comprises a core/shell structure,while in other embodiments, the Qdot is a core-only Qdot. ExemplaryQdots and methods for use and production are described in Rech-Genger etal., Quantum dots versus organic dyes as fluorescent labels. NatureMethods 2008 (9):763-775, incorporated herein in its entirety byreference for disclosure of fluorescent Qdots and organic dyes, andmethods of production and use of same).

In some embodiments, the carrier molecule is a water-soluble molecule.The term “water-soluble” is art-recognized and qualifies that an agentcan be dissolved in water to a certain degree, or, in other words, thata certain amount of the agent can be dissolved in a certain volume ofwater. For example, in some embodiments described here, a water-solublecarrier molecule is a carrier molecule that exhibits a solubility inwater at 25° C. and 1 ATM of more than 0.1 g/ml, more than 0.2 g/ml,more than 0.25 g/ml, more than 0.3 g/ml, more than 0.4 g/ml, more than0.5 g/ml, more than 0.6 g/ml, more than 0.7 g/ml, more than 0.8 g/ml,more than 0.9 g/ml, more than 1 g/ml, more than 1.1 g/ml, more than 1.2g/ml, more than 1.3 g/ml, more than 1.4 g/ml, more than 1.5 g/ml, morethan 1.6 g/ml, more than 1.7 g/ml, more than 1.8 g/ml, more than 1.9g/ml, more than 2 g/ml, more than 2.25 g/ml, more than 2.5 mg/ml, morethan 3 g/ml, more than 4 g/ml, more than 5 g/ml, more than 6 g/ml, morethan 7 g/ml, more than 8 g/ml, more than 9 g/ml, more than 10 mg/ml, ormore than 20 mg/ml.

In some embodiments, the carrier molecule is not water soluble. In somesuch embodiments, the carrier molecule is highly dispersible in waterand/or does not precipitate in aqueous solution under physiologicalconditions. In some embodiments, the diameter of the carrier molecule isless than 0.1 nm, less than 0.2 nm, less than 0.25 nm, less than 0.3 nm,less than 0.4 nm, less than 0.5 nm, less than 0.6 nm, less than 0.7 nm,less than 0.75 nm, less than 0.8 nm, less than 0.9 nm, less than 1 nm,less than 1.1 nm, less than 1.2 nm, less than 1.3 nm, less than 1.4 nm,less than 1.5 nm, less than 1.6 nm, less than 1.7 nm, less than 1.8 nm,less than 1.9 nm, less than 2 nm, less than 2.5 nm, less than 3 nm, lessthan 4 nm, less than 5 nm, less than 6 nm, less than 7 nm, less than 8nm, less than 9 nm, or less than 10 nm. In some embodiments, thediameter of the carrier molecule is less than 20 nm, less than 30 nm,less than 40 nm, less than 50 nm, less than 60 nm, less than 70 nm, lessthan 80 nm, less than 90 nm, less than 100 nm, less than 200 nm, lessthan 300 nm, less than 400 nm, less than 500 nm, less than 600 nm, lessthan 700 nm, less than 800 nm, less than 900 nm, or less than 1 μm.

In some embodiments, the non-covalent interaction is a non-covalent bondwith a dissociation constant K_(D) of 5 μM>K_(D)≧1 fM, for example, of100 nM>K_(D)≧1 pM, or of 100 nM>K_(D)≧100 fM. The term “dissociationconstant,” abbreviated as K_(D) herein, is art-recognized and refers toa specific type of equilibrium constant that measures the propensity ofa complex of associated molecules to separate (dissociate) reversiblyinto the separate molecules. The dissociation constant is the inverse ofthe association constant. For a general reaction A_(x)B_(y)

xA+yB, in which a complex A_(x)B_(y) breaks down into x A subunits and yB subunits, the dissociation constant is defined as

${K_{d} = \frac{\lbrack A\rbrack^{x} \times \lbrack B\rbrack^{y}}{\lbrack {A_{x}B_{y}} \rbrack}},$

where [A], [B], and [A_(x)B_(y)] are the concentrations of A, B, and thecomplex A_(x)B_(y), respectively. In some embodiments, a proteinmultimer is provided in which at least one of the protein monomers isconjugated to the carrier molecule via a non-covalent bond with adissociation constant 5 μM>KD>1 nM. In some embodiments, a proteinmultimer is provided in which at least one of the protein monomers isconjugated to the carrier molecule via a non-covalent bond with adissociation constant 5 μM>KD>1 μM. In some embodiments, a proteinmultimer is provided in which at least one of the protein monomers isconjugated to the carrier molecule via a non-covalent bond with adissociation constant of less than 2 fM, less than 5 fM, less than 10fM, less than 20 fM, less than 50 fM, less than 100 fM, less than 250fM, less than 500 fM, less than 1 pM, less than 2 pM, less than 5 pM,less than 10 pM, less than 20 pM, less than 50 pM, less than 100 pM,less than 250 pM, less than 500 pM, less than 1 nM, less than 5 nM, lessthan 10 nM, less than 20 nM, less than 50 nM, less than 100 nM, lessthan 250 nM, less than 500 nM, less than 600 nM, less than 700 nM, lessthan 800 nM, less than 900 nM, less than 1000 nM, less than 1500 nM,less than 2000 nM, less than 2500 nM, less than 3000 nM, less than 3500nM, less than 4000 nM, less than 4100 nM, less than 4200 nM, less than4300 nM, less than 4400 nM, less than 4500 nM, less than 4600 nM, lessthan 4700 nM, less than 4800 nM, less than 4900 nM, less than 5000 nM,more than 1 fM, more than 5 fM, more than 10 fM, more than 20 fM, morethan 25 fM, more than 50 fM, more than 100 fM, more than 200 fM, morethan 500 fM, more than 1 pM, more than 1 pM, more than 5 pM, more than10 pM, more than 20 pM, more than 25 pM, more than 50 pM, more than 100pM, more than 200 pM, more than 500 pM, more than 1 nM, more than 5 nM,more than 10 nM, more than 20 nM, more than 50 nM, more than 100 nM,more than 200 nM, more than 500 nM, more than 1000 nM, more than 2000nM, more than 2500 nM, more than 3000 nM, more than 3500 nM, more than4000 nM, more than 4100 nM, more than 4200 nM, more than 4300 nM, morethan 4400 nM, more than 4500 nM, more than 4600 nM, more than 4700 nM,more than 4800 nM, or more than 4900 nM, or any possible combination ofany of these values. For example, in some embodiments, a proteinmultimer is provided in which at least one of the protein monomers isconjugated to the carrier molecule via a non-covalent bond with adissociation constant of more than 1 pM and less than 10 nM, more than10 pM and less than 1000 nM, more than 100 pM and less than 500 nM, morethan 10 nM and less than 1000 nM, more than 10 nM and less than 100 nM,more than 100 nM and less than 4000 nM, more than 200 nM and less than3000 nM, more than 500 nM and less than 2000 nM, or more than 500 pM andless than 1000 nM.

In some embodiments, the carrier molecule or the protein, or both, areconjugated to a chelant moiety via a covalently bound linker. The term“linker,” as used herein, refers to a chemical structure between twomolecules or moieties or between a molecule and a moiety, thus linkingthe two. In some embodiments, the linker is covalently bound to bothlinked elements. In some embodiments, the linker is covalently bound toone, but not the other linked element. In some embodiments, the linkeris non-covalently bound to one or both elements. For example, in someembodiments, a linker is covalently bound to a carrier molecule and achelant moiety, while in other embodiments, a linker is covalently boundto a chelant moiety and non-covalently bound to a carrier molecule. Insome embodiments, the linker is about 2 Å, about 3 Å, about 4 Å, about 5Å, about 6 Å, about 7 Å, about 8 Å, about 9 Å, about 10 Å, about 11 Å,about 12 Å, about 13 Å, about 14 Å, about 15 Å, about 16 Å, about 17 Å,about 18 Å, about 19 Å, about 20 Å, about 21 Å, about 22 Å, about 23 Å,about 24 Å, about 25 Å, about 26 Å, about 27 Å, about 28 Å, about 29 Å,about 30 Å, about 35 Å, about 40 Å, about 45 Å, or about 50 Å long. Insome embodiments, the linker is longer than about 50 Å.

In some embodiments, NTA moieties are conjugated with a carriermolecule, for example, a PE molecule, using maleimide alkylationchemistry. For example, in some embodiments, PE is first reacted with anNHS-maleimide and the maleimido-PE subsequently with NTA-Cys (SH),resulting in the formation of a stable thio-ether. It is important tonote that the maleimido group readily hydrolyzes under certainconditions, which can limit the reproducibility and the degree ofconjugation.

In some embodiments, chelate moieties, for example, NTA moieties, areconjugated to a carrier molecule, for example, a PE molecule, with anoxime formation chemistry strategy. Using oxime chemistry has severaladvantages: i) efficient conjugation in the pH range 5-7; ii) goodstability under physiological conditions; and iii) efficient conjugationat low protein concentrations. An exemplary oxime chemistry forattachment of NTA moieties to a carrier is described in the followingscheme:

Exemplary oxime chemistry scheme.

The chemistry strategies provided herein, for example, the maleimide andoxime chemistry strategies described, are universally applicable togenerate MHC multimers, for example, multimers of MHC class I or classII as described herein. Additional maleimide and oxime chemistrystrategies will be apparent to those of skill in the art and it will beappreciated that the disclosure is not limited in this respect.

The use of oxime chemistry, also referred to herein as oxime ligation,has some advantages in the context of the preparation of largebioconjugates, for example, as its formation is compatible with otherfunctional groups often used, namely thiol-maleimide reactions or clickchemistry. Oxime formation benefits from the exclusive specificity andreactivity between the aminooxy function and the carbonyl group, sincethe nitrogen atom behaves as a weak base and as an excellentnucleophile. In general, aldehydes are substantially more reactive thanketones, mainly due to steric effects. The oxime ligation reaction canoccur in mild acidic conditions (pH 5) within 1 h but also atphysiological conditions (pH 7) in 10 h. The resulting imine bond iscovalent and stable under physiological conditions. In some embodiments,aminooxy-containing peptides are obtained by inserting the classicalfmoc-Dpr(Aoa)-OH residue at the last stage of solid phase peptidesynthesis. Incorporation of this residue is efficient (quantitativeyields) and does not require specialized conditions. Final TFAdeprotection of the peptide results in a free aminooxy function. Theincorporation of the aldehyde moiety on the biomolecule to bederivatized is achieved, in some embodiments, by employing acommercially available sulfo-SFB molecule, that reacts on lysines viasuccinimidyl ester reaction. One of the advantages of using oximechemistry over using a thiol-maleimide chemistry strategy is that oximeformation is not subjected to a hydrolysis or degradation reaction. Insome embodiments, oxime chemistry strategies lead to betterincorporation yields. Another benefit is that the functionalizedentities can be prepared and stored for several months before beingmixed together.

Oxime chemistry reactions, reagents, and reaction conditions are wellknown to those of skill in the art. Some reactions, reagents, andreaction conditions are described herein. Additional suitable reactions,reagents, and reaction conditions will be apparent to those of skill inthe art and it will be appreciated that this disclosure is not limitedin this respect. Suitable reactions, reagents, and reaction conditionsare described, for example, in Mathieu Galibert, Olivier Renaudet,Pascal Dumy, and Didier Boturyn. Angew. Chem. Int. Ed. 2011, 50, 1-5;Youhei Sohma and Stephen B. H. Kent. J. AM. CHEM. SOC. 2009, 131,16313-16318 9 16313; Anouk Dirksen and Philip E. Dawson. BioconjugateChem. 2008, 19, 2543-2548; Jenks, W. P. J. Am. Chem. Soc. 1959, 81,475-481; Rose, K. J. Am. Chem. Soc. 1994, 116, 30-33; Shao, J.; Tam, J.P. J. Am. Chem. Soc. 1995, 117, 3893-3899; Decostaire, I. P.; Lelie'vre,D.; Zhang, H.; Delmas, A. F. Tetrahedron Lett. 2006, 47, 7075-7060;Garanger, E.; Boturyn, D.; Renaudet, O.; Defrancq, E.; Dumy, P. J. Org.Chem. 2006, 71, 2402-2410; and Boturyn, D.; Coll, J. L.; Garanger, E.;Favrot, M. C.; Dumy, P. J. Am. Chem. Soc. 2004, 126, 5730-5739; theentire contents of each of which are incorporated herein by reference.

In some embodiments, a protein multimer is provided in which a pluralityof proteins is conjugated to a multivalent carrier molecule and whereinat least one of the proteins is conjugated to the carrier molecule via achelate complex bond.

The term “chelate complex,” as used herein, refers to a chemicalstructure that comprises two or more separate, non-covalent bindinginteractions between a polydentate (multiple bonded) molecule or moiety,also referred to as “chelant”, and a single central atom, for example, adivalent cation. The term “chelate complex bond,” accordingly, refers toa non-covalent bond between two or more chelants that form a chelatecomplex. In some embodiments, all chelants of a chelate complex are ofthe same structure. In other embodiments, a chelate complex is formed bychelants of different structures, for example, by a chelant comprising ahistidine residue and a chelant comprising an NTA residue. In someembodiments, the central atom is a divalent cation, for example, an Ni²⁺cation. Chelants, chelant moieties, and suitable central atoms are wellknown to those of skill in the art and the invention is not limited inthis respect.

In some embodiments, a protein multimer is provided in which a pluralityof MHC molecules is conjugated to a carrier molecule by a non-covalentbond as described herein, for example, a chelate complex bond or a bondof a K_(D) value as provided elsewhere herein.

The term “MHC molecule,” as used herein, refers to a protein encoded bythe major histocompatibility complex, and includes MHC class I and MHCclass II molecules. In some embodiments, the MHC molecule is an MHCclass I molecule and the MHC multimer is an MHC class I multimer. Insome embodiments, the MHC molecule is an MHC class II molecule and theMHC multimer is an MHC class II multimer.

In some embodiments, the MHC molecule is a human MHC molecule. Inhumans, MHC molecules are also referred to as HLA molecules. In someembodiments, the MHC molecule is an MHC molecule of a non-human mammal,for example, of a mouse, a rat, a rabbit, a non-human primate, a cat, adog, a goat, a cow, a sheep, a horse, or a pig.

MHC class-I molecules comprise one heavy chain type a that comprisesthree domains (α1, α2, and α3). In naturally occurring MHC class Imolecules, these domains are exposed to the extracellular space, and arelinked to the cellular membrane through a transmembrane region. The αchain of MHC class I molecules is associated with a molecule of β2microglobulin, which is not encoded by an MHC gene, but also includedwithin the scope of the term “MHC molecule”. MHC class II moleculescomprise two heavy chains, one type α and one type β, each of whichcomprises two domains: α1 and α2, β1 and β2, respectively. In naturallyoccurring MHC class II molecules, these domains are exposed to theextracellular space and are linked to the cellular membrane through atransmembrane region on each of the two chains.

In some embodiments, of this invention, an MHC multimer comprises agenetically engineered MHC molecule. In some embodiments, an MHCmolecule as provided herein comprises an extracellular domain of anaturally occurring MHC molecule, or a genetically engineered derivativethereof, but is devoid of all or part of the transmembrane domain ordomains. In some embodiments, MHC class II molecules are provided thatcomprise a leucine zipper in place of the transmembrane domain in orderto achieve dimerization of α and β chains. Genetically engineered MHCproteins, for example, MHC molecules lacking transmembrane domains, MHCmolecules comprising leucine zippers, single chain MHC molecules or MHCmolecules fused to an antigenic peptide, are also included in the scopeof the term “MHC molecule”. In some embodiments, the term “MHC molecule”refers to a complete molecule, for example, an MHC heavy chain type α(genetically engineered or not) that is associated with a molecule of β2microglobulin in the case of an MHC class I molecule, or an MHC heavychain type α (genetically engineered or not) that is associated with anMHC heavy chain type β (genetically engineered or not), for example, vialeucine zipper interaction. In some embodiments, the term “MHC molecule”refers to a single component of an MHC molecule, for example, to an MHCheavy chain (e.g. type α or type β, genetically engineered or not), orto a β2 microglobulin.

MHC molecules can bind antigenic peptides in a groove formed by the α1and α2 domains of MHC class I molecules or by the α1 and β1 domains ofMHC class II molecules. An MHC molecule that has bound an antigenicpeptide is referred to herein as a “peptide-loaded” MHC molecule,whereas an MHC molecule that has not bound an antigenic peptide isreferred to herein as an “empty” MHC molecule. The term “antigenicpeptide,” as used herein, refers to a peptide comprising a structurethat is recognized by the immune system of a subject. Non-limitingexamples of antigenic peptides are a peptide that is recognized by a Bor T-cell, e.g. via binding to a T-cell receptor, or a peptide thatbinds to an antibody or antibody fragment, or a peptide that stimulatesan immune response in a subject.

The term “monomeric MHC molecule,” “MHC monomer,” and “MHC moleculemonomer,” as used herein, refer to a single MHC molecule, for example,to a single MHC heavy chain, a single MHC heavy chain associated with aβ2 microglobulin, or a heterodimer of an MHC type α heavy chain and anMHC type β heavy chain. The term “MHC multimer,” as used herein, refersto a plurality of MHC molecules associated with each other, for example,via non-covalent interaction with a carrier molecule.

In some embodiments, the term “multimer” excludes dimers, but includestrimers, and multimers of four monomers (tetramers), or of more thanfour monomers (pentamers, hexamers, septamers, octamers, nonamers,decamers, etc.). In some embodiments, the term “multimer” excludesdimers and trimers, but includes multimers of four and more monomers.

Core Multimer Structure

Some aspects of this invention provide a multivalent chelant, thatcomprises a plurality of chelant moieties conjugated to a carriermolecule. Such multivalent chelants are useful for the generation ofreversible multimers, for example, of reversible protein multimers (e.g.MHC multimers), as described elsewhere herein.

The term “multivalent chelant molecule,” as used herein, refers to acarrier molecule comprising or conjugated to a plurality of chelantmoieties able to form chelate complex bonds with a plurality ofchelant-moiety comprising molecules. For example, a tetravalent chelantmolecule is a carrier molecule that is able to form chelate complexbonds to four molecules, for example, four MHC proteins comprising acompatible chelant moiety, thus forming a tetramer of the four moleculesheld together by chelate complex bonds. A compatible chelant moiety is achelant moiety able to form a chelate complex bond with the chelantmoiety of the carrier molecule. For example, an NTA moiety and aHistidine moiety are compatible chelant moieties, since they can form achelant complex bond in the presence of a divalent cation. In someembodiments, the multivalent chelant molecule can form a chelate complexbond with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, or 150 molecules comprisinga compatible chelant moiety. In some embodiments, the multivalentchelant molecule can form a chelate complex bond with more than 150compatible molecules.

In some embodiments, the carrier molecule is a water-soluble molecule asdescribed in more detail elsewhere herein. In some embodiments, thecarrier molecule is a non-water soluble molecule as described in moredetail elsewhere herein.

In some embodiments, the chelant moieties are tridentate or tetradentatechelant moieties. In some embodiments, the chelant moieties arenitrilotriacetic acid (NTA) moieties. In some embodiments, the chelantmoieties are histidine residues, for example, in the form of histidinetags. In some embodiments, the chelate moieties are iminodiacetic acid(IDA) moieties.

In some embodiments, the chelant moieties are covalently bound to thecarrier molecule via a linker, for example, via a linker describedherein. In some embodiments, each linker is bound to a single chelantmoiety (e.g., mono-NTA configuration). In some embodiments, two chelantmoieties are bound to a single linker (e.g., di-NTA configuration). Insome embodiments, four chelant moieties are bound to a single linker(e.g., tetra-NTA configuration).

Methods for Generation of Protein Multimers

In some embodiments, the invention provides methods for the generationof reversible protein multimers in which a plurality of proteins isconjugated to a carrier molecule via a chelate complex bond. In somesuch embodiments, the carrier molecule and the protein are conjugated tothe respective chelant moieties via covalent bond, for example, via acovalently bound linker. In such embodiments, the only non-covalent bondbetween carrier molecule and protein monomer is the chelate complexbond. In other embodiments, an additional non-covalent bond isintroduced between the carrier molecule and the monomeric protein, forexample, a binding molecule/ligand bond, such as a streptavidin/biotininteraction.

Some aspects of this invention provide methods for the generation ofprotein multimers, for example, of MHC protein multimers. In someembodiments, the method includes a step of contacting a monomericprotein, for example, an MHC molecule, that is conjugated to a chelantmoiety with a carrier molecule as provided herein, that is conjugated toa plurality of chelant moieties under conditions suitable for formationof a chelate complex between the chelant moieties conjugated to the MHCmolecule and the chelant moieties conjugated to the carrier molecule.

In some embodiments, a method for the generation of protein multimers,for example, MHC protein multimers is provided that includes contactinga protein molecule, for example, an MHC molecule, that is conjugated toa first chelant, for example, a His tag, with a ligand molecule, forexample, biotin, conjugated to a second chelant under conditionssuitable for formation of a chelate complex between the first and thesecond chelant. The resulting product is a monomeric protein conjugatedto a ligand via a non-covalent chelate complex bond. Such monomers canbe assembled to reversible multimers by contacting them with a carriermolecule conjugated to a plurality of ligand-binding molecules ormoieties, for example, streptavidin molecules. In some embodiments, amultivalent carrier molecule is generated by contacting a carriermolecule conjugated to a plurality of chelant moieties via non-covalentinteraction, for example, via biotin/streptavidin interaction. Forexample, in some embodiments, a carrier molecule comprising a pluralityof streptavidin moieties is contacted with a plurality of biotinmolecules that are conjugated to chelant moieties, for example, NTAmoieties, via a covalently bound linker as described herein.

In some embodiments, methods for the production of peptide-loaded MHCmultimers are provided. While stable, peptide-loaded MHC class Iproteins can be obtained by refolding of MHC class I heavy chains withpeptides of interest, recombinant MHC class II proteins are moredifficult to obtain. In some embodiments, recombinant MHC class IImolecules are produced in soluble form by insect expression systems,such as Drosophila S2 cells or baculovirus and sf9 cells. With very fewexceptions, deletion of the transmembrane (TM) domains of the α and βchains in MHC class II molecules results in the dissociation of the twosubunit chains. In some embodiments, chain pairing is re-established byaddition of leucine zippers. In some embodiments, empty MHC class IImolecules are first isolated and then loaded with an antigenic peptideof interest. Such peptide-loaded MHC class II molecules can then beisolated and used in the production of MHC protein multimers. In someembodiments, for example, in embodiments where the binding interactionbetween the peptide of interest and an MHC class II molecule is of lowstrength, peptides can be fused to the N-terminus of the β chain via aflexible linker. Such fusions of MHC class II chains and antigenicpeptides, resulting in the production of recombinant, peptide-loaded MHCmolecules, are well known to those of skill in the art.

In some embodiments, a chelant moiety, for example, a His tag, is addedat the C-terminus of an MHC chain by recombinant addition of a fusionpeptide comprising a chelant moiety, for example, in the form of a Histag as described herein. In some embodiments, for example, in someembodiments, in which the peptide-loaded MHC monomer is isolated via amethod using a tag attached to the antigenic peptide, a chelant moietyis attached to the isolated, peptide-loaded MHC molecule afterisolation. Methods for post-synthesis or post-isolation of chelants toisolated proteins are known to those of skill in the art and exemplarymethods are described herein.

While some MHC molecules are instable without a bound antigenic peptide,in some embodiments, the MHC molecule is sufficiently stable withoutpeptide cargo (e.g. HLA DRB1*0101 or DRB1*0401) to allow the productionof empty MHC molecules and MHC multimers, e.g., MHC molecules ormultimers that are not loaded with an antigenic peptide. In some suchembodiments, the MHC monomer is loaded after isolation or purificationwith the peptide of interest. In some embodiments, the MHC monomer isfirst incorporated into a reversible MHC multimer as provided herein andsubsequently loaded with a peptide of interest. The efficiency ofpeptide loading strongly depends on its binding strength to therespective MHC molecule. If the binding is below a critical threshold,peptide loading is inefficient and the resulting complexes are oflimited stability, both physically and conformationally.

Some embodiments of the invention provide methods for the generation ofMHC molecules and multimers that are loaded with an antigenic peptide.In some embodiments, methods and reagents for the production ofpeptide-loaded MHC class II molecules are provided. The production ofpeptide-loaded MHC class II molecules is technically difficult, based onthe instability of engineered MHC class II heterodimers comprising α andβ heavy chains lacking a transmembrane domain, and, in many instances,the low affinity binding interactions between the MHC class II heavychains and the antigenic peptide. As a result, populations ofpeptide-loaded MHC class II molecules produced with conventional methodsare often heterogeneous in that a significant portion of MHC class IImolecules are not or not correctly peptide-loaded. MHC class IImultimers produced from such heterogeneous populations of MHC class IImolecules often show poor staining performance, great batch-to-batchvariability in staining efficiency, and some specific peptide-loaded MHCclass II multimers are difficult or impossible to obtain withconventional methods.

Some aspects of this invention provide methods addressing these problemsin the production of MHC class II molecules and multimers. For example,some aspects of this invention provide methods and reagents for thegeneration of peptide-loaded MHC molecule, for example, MHC class IImolecules, that include the use of a tag conjugated to the antigenicpeptide. In some embodiments MHC molecules that have bound a taggedantigenic peptide are isolated and/or purified by a method that can becarried out under non-denaturing conditions, for example, by certainchromatography methods (e.g., affinity chromatography or ion exchangechromatography). In some embodiments, the tag conjugated to theantigenic peptide can be removed, for example, by cleaving a linker thatconnects the tag to the antigenic peptide, and methods for tag removalfrom tagged peptide-loaded MHC molecules, for example, MHC class IImolecules, are also provided herein.

In some embodiments, the antigenic peptide of interest is conjugated toa tag. In some embodiment, the tag is a peptide tag, for example, apeptide tag that is N-terminally or C-terminally fused to the antigenicpeptide. In some embodiments, the tag is an affinity tag that allows forthe isolation of correctly loaded MHC class II molecules by affinitychromatography. Affinity tags are well known to those of skill in theart and examples of peptide tags include, but are not limited to, biotincarboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags,FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred toas histidine tags or His-tags, maltose binding protein (MBP)-tags,nus-tags, glutathione-S-transferase (GST)-tags, green fluorescentprotein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1,Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5 tags, andSBP-tags. In some embodiments, the tag is a biotin tag or a biotinvariant tag, for example, desthiobiotin (DTB). DTB is a biotin variantthat binds about 1×10⁶-fold weaker to streptavidin than biotin. DTB isreadily displaced by free biotin, allowing gentle affinity purificationbased on the reversible DTB-streptavidin conjugation. Conjugationpartners similar to streptavidin can also be employed, for example,StreptActin, a mutant of streptavidin. Strep tags, which are peptidicbiotin analog, bind also to StreptActin. Desthiobiotin, biotin,streptavidin, StreptActin, strep tags and derivatives of these reagents,as well as methods for the use of these reagents in protein and peptidepurification are well known to those of skill in the art. Some methodssuitable according to aspects of this invention are described herein,and additional suitable methods will be apparent to those of skill, forexample, as described in Howarth M, Chinnapen D J, Gerrow K, DorresteinP C, Grandy M R, Kelleher N L, El-Husseini A, Ting A Y. A monovalentstreptavidin with a single femtomolar biotin binding site. Nat. Methods.2006; 3:267-73; Hirsch J D, Eslamizar L, Filanoski B J, Malekzadeh N,Haugland R P, Beechem J M, Haugland R P. Easily reversible desthiobiotinbinding to streptavidin, avidin, and other biotin-binding proteins: usesfor protein labeling, detection, and isolation. Anal Biochem. 2002;308:343-57; Lichty J J, Malecki J L, Agnew H D, Michelson-Horowitz D J,Tan S. Comparison of affinity tags for protein purification. ProteinExpr Purif. 2005; 41:98-105; Wu S C, Wong S L. Development of anenzymatic method for site-specific incorporation of desthiobiotin torecombinant proteins in vitro. Anal Biochem. 2004; 331(2):340-8; MaierT, Drapal N, Thanbichler M, Bock A. Strep-tag II affinity purification:an approach to study intermediates of metalloenzyme biosynthesis. AnalBiochem. 1998; 259:68-73; Chen R, Folarin N, Ho V H, McNally D, DarlingD, Farzaneh F, Slater N K. Affinity recovery of lentivirus bydiaminopelargonic acid mediated desthiobiotin labeling. J Chromatogr BAnalyt Technol Biomed Life Sci. 2010; 878:1939-45; Gloeckner C J, BoldtK, Schumacher A, Roepman R, Ueffing M. A novel tandem affinitypurification strategy for the efficient isolation and characterisationof native protein complexes. Proteomics. 2007; 7:4228-34; Cass B, Pham PL, Kamen A, Durocher Y. Purification of recombinant proteins frommammalian cell culture using a generic double-affinity chromatographyscheme. Protein Expr Purif. 2005 March; 40(1):77-85; Korndörfer I P,Skerra A. Improved affinity of engineered streptavidin for the Strep-tagII peptide is due to a fixed open conformation of the lid-like loop atthe binding site. Protein Sci. 2002; 11:883-93; the entire contents ofeach of which are incorporated herein by reference.

Sequences of peptide tags useful in some embodiments of this invention,for example, the tags described herein, are well known to those of skillin the art, and exemplary tags are described, for example, in Kimple, M.E., and Sondek, J. Overview of affinity tags for protein purification.Curr Protoc Protein Sci. 2004 September; Chapter 9:Unit 9.9,incorporated in its entirety herein for disclosure of affinity tags.Those of skill in the art will appreciate that the invention is notlimited in this respect.

Some aspects of this invention provide tagged MHC class II bindingantigenic peptides and methods of using such peptides. In someembodiments, a tag conjugated to an antigenic peptide is useful for theisolation of the tagged peptide, either alone or when bound to an MHCclass II molecule. Methods for isolating tagged peptides are well knownto those of skill in the art and include, for example, affinitychromatography and ion exchange chromatography.

In some embodiments, an MHC class II binding peptide is provided or usedthat is conjugated to a tag. In some embodiments, the tag is an acidictag. In some embodiments, the acidic tag is an acidic peptide tag, forexample, a peptide tag comprising a sequence of 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 16, 17, 18, 19, 20, or more contiguous acidic aminoacid residues. In some embodiments, the acidic amino acid residuesglutamic acid (Glu, E) or aspartic acid (Asp, D) residues. In someembodiments, the antigenic peptide is conjugated to an acidic tag thatallows for the isolation of correctly loaded MHC class II molecules byanion exchange chromatography. In some embodiments, the tag is a tagcomprising a sequence of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Aspresidues, for example, a pY-D4 tag (phosphor-tyrosine followed by fourAsp residues), a pY-D5, pY-D6, pY-D7, pY-D8, pY-D9, or pY-D10 tag. Insome embodiments, the acidic tag is a tag comprising a sequence of 2, 3,4, 5, 6, 7, 8, 9, 10, or more Glu residues, for example, a pY-E6(phosphor-tyrosine followed by six Glu), pY-E7, pY-E8, pY-E9, or apY-E10 tag. In some embodiments, the acidic tag is an acidic detectablelabel, for example, an acidic fluorophore. In some embodiments, theacidic fluorophore is a cyanine dye tag, for example, a Cy5 tag, or aCy5.5 tag. Other acidic tags suitable for peptide or protein isolationand/or purification are well known to those of skill in the art and theinvention is not limited in this respect.

In some embodiments, tagged MHC class II binding peptides are providedthat are reversibly tagged, e.g., that are tagged with a structure thatcan be cleaved, resulting in a release of the conjugated tag from thepeptide. Methods of using such reversibly tagged peptides are alsoprovided. In some embodiments, the tag is conjugated to the peptide viaa cleavable linker. In some embodiments, the linker is a photocleavablelinker. In some embodiments, the linker is a 2-nitro-phenyl-β-Ala (NPβA)linker. In some embodiments, the linker is a peptide linker thatcomprises an amino acid sequence that can be cleaved by a protease or bya chemical. In some embodiments, the tagged peptide is a peptideconjugated to a part of a cleavable linker that remains after cleavageof the linker.

One advantage of a cleavable linker is that after isolation of thepeptide-loaded MHC class II molecule with a method relying on the tag,the tag can be removed. Cleavage of a cleavable linker will, in someembodiments, leave part of the linker conjugated to the MHC class IIbinding peptide. However, some linkers can be designed in a manner thatwill result in complete removal of the linker from the MHC-class IIbinding peptide.

Tag removal by linker cleavage is particularly useful in embodiments,where the tag interferes or is suspected to interfere with the bindingof the peptide-loaded MHC class II molecule to its target T-cellreceptor. Exemplary cleavable linkers are described in more detailelsewhere herein. Additional cleavable linkers are known to those ofskill in the art and the invention is not limited in this respect. Insome embodiments, the cleavable linker is a photocleavable linker.Photocleavable linkers can be cleaved by irradiation with UV light.

Photocleavable linkers are described herein and additionalphotocleavable linkers are well known in the art. See, for example,Pandori M W, Hobson D A, Olejnik J, Krzymanska-Olejnik E, Rothschild KJ, Palmer A A, Phillips T J, Sano T., Photochemical control of theinfectivity of adenoviral vectors using a novel photocleavablebiotinylation reagent. Chem. Biol. 2002 May; 9(5):567-73; Hahner S,Olejnik J, Lüdemann H C, Krzymañska-Olejnik E, Hillenkamp F, RothschildK J. Matrix-assisted laser desorption/ionization mass spectrometry ofDNA using photocleavable biotin. Biomol Eng. 1999 Dec. 31;16(1-4):127-33; Olejnik J, Lüdemann H C, Krzymañska-Olejnik E,Berkenkamp S, Hillenkamp F, Rothschild K J. Photocleavable peptide-DNAconjugates: synthesis and applications to DNA analysis using MALDI-MS.Nucleic Acids Res. 1999 Dec. 1; 27(23):4626-31; Olejnik J,Krzymañska-Olejnik E, Rothschild K J. Photocleavable aminotagphosphoramidites for 5′-termini DNA/RNA labeling. Nucleic Acids Res.1998 Aug. 1; 26(15):3572-6. Olejnik J, Krzymañska-Olejnik E, RothschildK J. Photocleavable affinity tags for isolation and detection ofbiomolecules. Methods Enzymol. 1998; 291:135-54; Olejnik J, Sonar S,Krzymañska-Olejnik E, Rothschild K J. Photocleavable biotin derivatives:a versatile approach for the isolation of biomolecules. Proc Natl AcadSci USA. 1995 Aug. 1; 92(16):7590-4; all references incorporated hereinby reference for disclosure of photocleavable linkers). In someembodiments, a peptide tagged via a cleavable linker is referred to as areversibly tagged peptide.

In some embodiments, an MHC class II molecule loaded with a reversiblytagged peptide is isolated, for example, by affinity or ion exchangechromatography, and the tag is removed after isolation by cleavage ofthe linker. In some such embodiments, the MHC class II molecule loadedwith a now untagged peptide is then isolated from the cleaved tag, forexample, by size fractionation.

In some embodiments, an MHC molecule, for example, an MHC class IImolecule conjugated to a chelant moiety is loaded with a tagged peptide.In some embodiments, the peptide tag is an acidic tag, as providedherein, and the chelant moiety is comprised in a peptide tag, forexample, a His tag, as provided herein. For example, in someembodiments, an MHC class II molecule is provided that comprises a heavychain conjugated to a His tag, for example, by C-terminal fusion, andthat is loaded with a tagged antigenic peptide, for example, anantigenic peptide conjugated to an acidic tag, for example, a Cy5.5 tag)via a photocleavable linker. In some embodiments, a correctlypeptide-loaded MHC class II molecule is isolated by anion exchangechromatography, the tag is cleaved from the antigenic peptide, forexample, by UV irradiation, and the peptide-loaded MHC class II moleculethus generated is then assembled into an MHC multimer as describedherein.

In some embodiments, an MHC molecule, for example, an MHC moleculeloaded with an antigenic peptide is conjugated with a chelant moiety ora peptide tag after production of the MHC molecule or after loading theMHC molecule with the antigenic peptide, or after cleavage of a tag, ifpresent, from a reversibly tagged antigenic peptide bound to the MHCmolecule. In some embodiments, such post-production conjugation, forexample, to a heavy chain of a peptide-loaded MHC class II molecule, iscarried out by site-specific alkylation or a sortase-mediatedtranspeptidation reaction. In some embodiments, the antigenic peptide istagged with a His tag, the tag is cleaved after peptide-loading, and aHis tag is subsequently appended to a heavy chain of the MHC molecule.Using such “tag exchange” strategies, incompatible tags can be usedsubsequently, or the same tag can be employed at different positions.Methods for post-synthesis addition of tags to peptides and proteins arewell known to those of skill in the art and include, but are not limitedto, biotinylation and sortase-mediated protein labeling (for the lattersee Popp et al., Site-Specific Protein Labeling via Sortase-MediatedTranspeptidation Curr. Protoc. Protein Sci. 56:15.3.1-15.3.9; 2009,incorporated herein by reference in its entirety for disclosure ofsortase-mediated transpeptidation reactions).

To give a non-limiting example of an embodiment employing such atag-exchange strategy: in some embodiments, an MHC class II moleculecomprising a sortase target sequence is loaded with a His-tagged MHCclass II-binding peptide and peptide-loaded MHC class II molecules areisolated form free peptide and empty MHC class II molecules by affinitychromatography and, optionally, size fractionation. After isolation, theHis-tag is cleaved off the MHC class II binding peptide and, optionally,the tag-free MHC class II molecule loaded with the peptide is separatedfrom the cleaved-off tag. In some such embodiments, the heavy chain ofthe MHC class II molecule is then tagged by performing asortase-mediated transpeptidation reaction. In some such embodiments,the tag appended to the MHC heavy chain is a peptide tag. In someembodiments, the sortase-appended tag is a His-tag, thus effectivelyyielding MHC class II molecules in which the His tag was moved from theantigenic peptide to a heavy chain.

In some embodiments that include a charged tag, for example an acidictag, that is conjugated to an MHC class II binding peptide, apeptide-loaded MHC class II molecule is isolated by ion exchangechromatography. In some embodiments including an acidic tag, anionexchange chromatography is used to isolate the tagged peptide or an MHCclass II molecule loaded with the tagged peptide.

Other methods of producing peptide-loaded MHC multimers are known in theart (for example, see Altman et al., Science 274:94 96, 1996; Dunbar etal., Curr. Biol. 8:413 416, 1998; Crawford et al., Immunity 8:675 682,1998). In all embodiments, non-denaturing conditions are preferredduring isolation of empty and peptide-loaded MHC class II molecules.

Some aspects of this invention provide methods and reagents for thegeneration of “empty” MHC class II molecules. The term “empty” in thecontext of MHC class II molecules signifies that the MHC molecules arenot loaded with an antigenic, MHC class II-binding peptide. Such emptyMHC class II molecules are often instable and conventional methods ofhigh-affinity antibody-mediated isolation are typically unsuitable forthe preparation of such empty MHC class II molecules because of thedenaturing conditions used in such methods.

Some aspects of this invention provide methods that allow gentlepurification of fragile “empty” (without nominal peptide cargo) Histagged MHC II molecules by affinity chromatography on Ni2+nitrilotriacetic acid (NTA) columns. In some embodiments of thisinvention, methods are provided that allow for the isolation of emptyMHC class II molecules that retain the correct folding and dimerizationproperties. In some embodiments, such methods include a step of tagginga heavy chain comprised in an MHC class II molecule. In someembodiments, the tag is a peptide tag that can be used for isolation ofthe tagged protein by affinity chromatography or ion exchangechromatography. In some embodiments, the tag is a His tag, for example,a tag that comprises 3-12 Histidine residues. In some embodiments, MHCclass II molecules comprising a His-tag labeled heavy chain are isolatedby affinity chromatography. In some embodiments, the affinitychromatography uses an Ni2+-NTA resin. Methods for affinitychromatography for the isolation of tagged peptides and proteins arewell known to those of skill in the art and it will be appreciated bythose of skill that the invention is not limited in this respect.

Empty MHC multimers are useful in that they allow for standardizedproduction of a core reagent that can then be customized for a specificapplication by loading a specific antigenic peptide of interest onto themultimer.

In some embodiments, a chelant moiety is conjugated to an isolatedprotein after the protein has been synthesized, for example,post-translationally, or after in situ synthesis, for example, afterFmoc synthesis. In some such embodiments, the chelant moiety isconjugated to the protein, for example, the MHC molecule, for example,to a heavy chain of the MHC molecule by chemical or enzymaticmodification. Methods for post-synthesis addition of tags to peptidesand proteins are well known to those of skill in the art and include,but are not limited to, biotinylation and sortase-mediated proteinlabeling (for the latter see Popp et al., Site-Specific Protein Labelingvia Sortase-Mediated Transpeptidation Curr. Protoc. Protein Sci.56:15.3.1-15.3.9; 2009, incorporated herein by reference in its entiretyfor disclosure of sortase-mediated transpeptidation reactions).

Post-isolation addition of a chelant moiety is particularly useful inembodiments, where a chelant moiety would interfere with a synthesis orpurification step, for example, in embodiments, where an MHC molecule isloaded with an antigenic peptide that comprises a chelant tag and thetag is used for isolation of peptide-loaded MHC molecules. In some suchembodiments, the peptide tag can be cleaved from the antigenic peptideand a chelant tag can be added subsequently to the MHC molecule, forexample, to an MHC heavy chain by methods well known to those of skillin the art.

Cell Staining and Detection Methods

Some aspects of this invention provide methods for the staining,detection, and/or isolation of cells using a reversible proteinmultimer, for example, a reversible MHC multimer, as described herein.In some embodiments, the method comprises contacting a population ofcells with a protein multimer, for example, an MHC multimer providedherein. In some embodiments, the multimer comprises a detectable label,for example, a fluorophore, either as the carrier molecule or conjugatedto the multimer.

The term “detectable label,” as used herein, refers to a molecule ormoiety that can be detected, for example, by performing an assay knownto those of skill in the art for its detection. A detectable label,accordingly, may be, for example, (i) an isotopic label (e.g., aradioactive or heavy isotope, including, but not limited to, 2H, 3H,13C, 14C, 15N, 31P, 32P, 35S, 67Ga, 99mTc (Tc-99m), 111In, 123I, 125I,169Yb, and 186Re), (ii) an affinity label (e.g., an antibody or antibodyfragment, an epitope, a ligand or a ligand-binding agent) (iii) andenzymatic label that produce detectable agents when contacted with asubstrate (e.g., a horseradish peroxidase or a luciferase); (iii) a dye,(e.g., a colored, luminescent, phosphorescent, or fluorescent molecule,such as a chemical compound or protein). Fluorophores, for example,fluorescent dyes and proteins, are of particular use for embodiments ofthis invention that involve detection or isolation of living cells. Afluorophore is a molecule or moiety that absorbs light of a specificwavelength and then re-emits light at a different specific wavelength,thus causing the molecule of moiety to be fluorescent. Other detectablelabels are known to those of skill in the art and the invention is notlimited in this respect. It will be appreciated that a detectable labelmay be incorporated into any part of the multimeric structure and in anymanner that does not interfere with the stability or the function, forexample, the binding activity of the multimer.

In some embodiments, the method includes a step of detecting themultimer bound to a cell, for example, to a surface receptor (e.g., aT-cell receptor) of a cell. The method performed to detect the multimerdepends, of course, on the nature of the detectable label comprised inthe multimer. For example, in some embodiments, where the detectablelabel is a fluorophore, suitable methods for detection are fluorescencemicroscopy, cytometry, or FACS.

In some embodiments, the method comprises a step of quantifying thenumber of detected cells, for example, quantifying the number of T cellsexpressing a specific TCR in a cell population, for example, in a cellpopulation obtained from a subject. In some embodiments, the quantity ofcells, for example, of T-cells expressing a specific TCR, is compared toa reference quantity. In some embodiments, the comparison of thequantity of T-cells expressing a specific TCR in a subject to areference quantity is used to determine an immune reaction in thesubject. In some embodiments, the reference quantity is a quantitymeasured or expected in a healthy subject or in healthy subjects, or aquantity measured in the subject prior to a clinical intervention, forexample, prior to a vaccination, and a quantity in the subject that ishigher than the reference is indicative of an immune response in thesubject, whereas a quantity in the subject that is lower than thereference is indicative of depletion of a specific T-cell population.

Accordingly, MHC multimers as provided herein are useful, for example,for monitoring immune responses in subjects, either in response to aclinical intervention, for example, a vaccination, or as a result of adisease or condition, for example, a hyperproliferative disease in thesubject. In some embodiments, the clinical intervention is a vaccinationagainst a tumor antigen. In some embodiments, the vaccination is avaccination administered after surgical removal of a tumor expressingthe tumor antigen. In some embodiments, the clinical intervention is anintervention aimed to suppress a function of the immune system, forexample, by depleting a specific population of T-cells. In someembodiments, the subject is a subject having an autoimmune disease.

In some embodiments, the detection method further comprises a step ofreleasing the chelate complex bond comprised in the multimer employed,for example, by withdrawing the cation of the complex bond, or bycontacting the multimers with an agent able to displace a chelantforming the chelate complex bond.

T Cell Staining Methods

Interactions of T cell antigen receptors (TCRs) with MHC-peptidemonomers are characterized by micromolar dissociation constants (K_(D))and half-lives in the range of seconds. The coordinate binding of CD8 toTCR-associated MHC class I-peptide complexes can considerably strengthenthe binding interaction. According to some aspects of this invention,the use of MHC-peptide monomers that are conjugated to reversiblemultimers substantially increases the overall binding avidity anddecreases the dissociation rate to half-lives in the order of hours.Accordingly, the use of MHC multimers as provided herein allows for theefficient staining, detection, and/or isolation of T cells bearingspecific TCRs, for example, by fluorescent microscopy, flow cytometry,fluorescence activated cell sorting (FACS), or magnetic-activated cellsorting (MACS).

In some embodiments, MHC multimers and methods are provided that areuseful for the staining of CD8⁺ T-cells. CD8 interacts preferentiallywith MHC class I molecules. Accordingly, MHC multimers useful forstaining of CD8⁺ T-cells are preferentially MHC class I multimers. CD8undergoes differentiation- and activation-dependent changes in theglycosylation and sialylation of its 0 chain, which can profoundlyaffect cognate and non-cognate MHC class I-peptide binding. Non-cognateCD8 binding to MHC class I-peptide multimers has been reported toincrease non-specific multimer binding. Accordingly, in someembodiments, multimers are provided that contain the CD8 bindingweakening mutation A245V in the MHC a3 domain. In some embodiments, anMHC class I multimer as provided herein (A245V mutated or not) is usedin a staining procedure at a concentration of about 5-30 nM (about 2.5to 15 μg/ml). At this concentration, non-specific staining is generallylow.

Importantly, binding of MHC class I molecules to T-cell receptors canelicit T cell activation events, such as intracellular calciummobilization, diverse tyrosine phosphorylation and endocytosis of MHCclass I-peptide engaged TCR/CD8. For example, MHC class I-peptidecomplex driven cell activation can induce death of effector cytotoxicT-cells (CTLs) via FasL-dependent apoptosis or severe mitochondrialdamage. This can lead to changes in T-cell populations that arecontacted with MHC multimers, for example, for cell staining, detection,or isolation, for example, by selective depletion of stained T-cells. Insome circumstances, TCR-activation-mediated cell depletion can renderisolation of a non-activated T-cell population impossible. Some aspectsof this invention provide reversible MHC multimers and methods for theiruse that avoid this problem by minimizing the time of high-avidityMHC/TCR interaction by re-monomerizing the multimers, thus minimizingundesired TCR activation-mediated effects on stained cells. However, itwill be appreciated by those of skill in the art, that the reversibleMHC multimers provided herein can also be used in methods that do notinclude a chelate complex bond release step, thus employing thereversible multimers in the manner conventional multimers would beemployed. Accordingly, in some embodiments, methods are provided thatexploit MHC multimer TCR activation to deplete specific T-cellpopulations by MHC-mediated TCR activation. In some such embodiments,reversible MHC multimers as provided herein are used to eradicateantigen-specific CD8+ CTLs.

MHC class II multimer binding to CD4+ T-cells can also lead to T-cellactivation and death, for example, of CD4+ effector cells. Accordingly,reversible MHC class II multimers are useful in the staining of CD4+T-cells and in the isolation of minimally manipulated or activated CD4+T-cells.

Some aspects of this invention provide reversible MHC multimers andmethods for the use of such multimers to analyze the state of activationor differentiation of T-cells, for example, CD8+ and/or CD4+ T-cells. Aswill be appreciated by those of skill in the art, homogenous populationsof MHC multimers of defined structure are preferable over heterogeneousMHC multimer populations. In some embodiments, homogenous populations ofMHC multimers, e.g. of dimers, trimers, tetramers, pentamers, hexamers,or decamers, are provided for use in such methods. In some embodiments,the MHC multimers comprise linkers of defined length and flexibility. Insome embodiments, the MHC multimers comprise chelant groups in definedconfigurations, for example, in mono- di- or tetra-chelantconfiguration. In contrast to heterogeneous multimers, binding studieswith defined, homogenous populations of multimers can revealdifferentiation- and activation-dependent differences, for example,differentiation- and activation-dependent changes in glycosylation andsialylation of T cell surface molecules involved in antigen recognitionof the cells under study which can affect, for example, CD8participation in MHC class I molecule binding and/or aggregation of TCRand CD8.

It will be apparent to those of skill in the art that the MHC multimersprovided herein can be employed alone or in combination with otherbinding and/or staining agents. For example, in some embodiments, MHCmultimers provided herein are used to stain T-cells in combination withstaining the cells for an additional antigen, for example, with astaining for intracellular cytokine staining.

In some embodiments, reversible MHC class I-peptide multimers, areprovided that comprise a mutation in the α3 domain. In some embodiments,the mutation is a mutation that ablate CD8 binding, e.g. a D227K, T228Ain human MHC and D227K, Q226A in mouse MHC molecules. In someembodiments, methods are provided that use such CD8 binding-deficientMHC multimers to stain, detect, and/or isolate CD8-independent T cells,which typically express high affinity TCRs.

For example, in some embodiments, CD8 binding-deficient MHC multimersare provided for the staining, detection, and/or isolation of CD8+ Tcells expressing high-affinity TCRs specific for tumor antigens, forexample, for MELAN-A/Mart-1, gp100, or tyrosinase. It is known to thoseof skill in the art that such tumor-antigen specific T-cell tend toexpress low affinity TCRs and that infrequent CD8+ T cells specific fortumor antigens expressing high affinity TCRs efficiently kill tumorcells. In some embodiments, the use of a reversible MHC class I multimeras provided herein enables efficient identification and isolation ofsuch rare cells with no or only minimal TCR activation, thus allowingfor the isolation of native T-cell populations that cannot be isolatedwith conventional MHC class I multimers.

Further, in some embodiments, CD8 binding-deficient multimers are usedto selectively induce FasL (CD95L) expression, resulting in apoptosis ofantigen-specific CTLs.

Conditions and protocols for staining, detection, and isolation of cellsusing multimers are well known to those of skill in the art. In general,methods for the use of conventional MHC multimers are applicable to thereversible MHC multimers as provided herein, modified, whereappropriate, to include a step of chelate complex bond release, asdescribed in more detail elsewhere herein.

Staining with reversible MHC multimers can be performed through a widerange of temperatures. In some embodiments, staining is performed at atemperature between 0-37° C.

In some embodiments, MHC multimer staining is performed at 37° C. Whilestaining at 37° C. is rapid, and efficient staining of CD4+ T cells withreversible MHC class II multimers is often observed upon incubations at37° C. for extended periods of time, reversible MHC class I multimersefficiently effect TCR activation at this temperature.

In some embodiments, staining is performed at 0-4° C. It will beappreciated by those of skill, that MHC multimer binding at lowtemperatures (e.g., 0-4° C.) tends to be slow, necessitating extendedperiods of time for staining as compared to staining at highertemperatures. In some embodiments, staining with MHC multimers isperformed at ambient temperature, e.g. at 20-30° C., preferably at22-25° C. In some embodiments, MHC staining is performed in the presenceof EDTA (e.g., 5 mM) and/or sodium azide (e.g., 0.02%) to inhibit cellactivation. Under these conditions multimer binding is rapid. In someembodiments, staining is performed for about 10 minutes, about 15minutes, about 20 minutes, about 25 minutes, about 30 minutes, or about20-45 minutes. In some embodiments, for example, in some embodimentsusing reversible MHC class II multimers, staining is performed for30-120 min. Under these conditions, cognate MHC class II complexesbinding to TCR (and CD4) are internalized and accumulate over time.

Multimer concentration is an important factor in achieving maximumstaining efficiency, and, while exemplary MHC multimer concentrationsare provided herein, it will be appreciated by those of skill that it ispreferable to test a range of concentrations, for example, in the rangeof about 5-50 nM (about 2.5-25 μg/ml), or, in the case of low affinitybinding, in higher concentration ranges, for example, in the range ofabout 5-100 nM (about 2.5-50 μg/ml).

In some embodiments in which a cell is contacted with an MHC multimer,for example, with an MHC class II multimer as provided herein, bindingof the MHC multimer to the cell is facilitated by desialylation of thecell. Desialylation is a process by which sialyl groups on the cellsurface are removed or modified. Methods and reagents for desialylatinga cell are described in detail elsewhere herein and additional methodsare well known to those of skill in the art. For example, in someembodiments, a cell is contacted with a desialylating agent in order toachieve desialylation. Desialylating agents are, in some embodiments,enzymes, while, in other embodiments, chemicals are used to effectdesialylation. Enzymes known to desialylate cell surfaces are, forexample, neuraminidases. Methods and conditions suitable fordesialylation of cells by contacting them with a neuraminidase are wellknown to those of skill in the art.

In some embodiments, the cells are pre-treated with neuraminidase underconditions suitable to achieve desialylation of the cells (e.g.,treatment with 0.03μ/ml for 30 min at 37° C.).

In some embodiments, staining is increased by inhibiting TCR downmodulation with the protein kinase inhibitor dasatinhib. In someembodiments, scarce antigen-specific cells can be enriched bycombination of fluorescence-based methods as described herein with anon-fluorescent-based isolation method, for example, with MACS usingmagnetic beads coated with an antibody against an epitope of the carriermolecule.

Isolation of Cells with Reversible Multimers

In some embodiments, methods for the use of multimers as describedherein for isolating specific cells or cell populations are provided. Ingeneral, useful multimers for isolation methods comprise a detectablelabel and the methods include a step of staining the target cellpopulation as described in more detail elsewhere herein. In someembodiments including the isolation of cells, a method of cellseparation is employed that allows for the enrichment or the isolationof homogenous populations of cells based on the cells binding theemployed multimer, for example, the employed MHC multimer. Such methodsare well known in the art and include, for example, FACS and MACS.

In some embodiments, the method of isolating cells with a reversiblemultimer further includes a step of releasing the chelate bond comprisedin the multimer. In some embodiments, this step includes withdrawal ofthe central ion, for example, the central divalent cation, from thechelate complex. Method for ion withdrawal are well known in the art andinclude, in some embodiments, washing the cells with a solution thatdoes not contain a significant amount of the divalent cation, or with asolution that comprises an agent that sequesters the divalent cation. Insome embodiments, the step of chelate complex bond release includescontacting the chelate complex bond with an agent that displaces achelant from the chelate complex bond. For example, if the chelatecomplex bond is formed by an NTA chelant and a histidine residue, thechelate complex bond can be released by contacting it with an imidazoleresidue, for example, with free imidazole. Imidazole is able to displacea chelant, in this case, the histidine chelant from the complex bond,thus releasing the chelate complex bond of the multimer. The result ofthis release is the re-monomerization of the proteins comprised in themultimer, for example, of MHC proteins in an MHC multimer.

In some embodiments, re-monomerization of the multimer after stainingand/or isolation avoids detrimental effects on the cells and, inembodiments, where the cells are rare and/or sensitive to detrimentaleffects of staining, allows for efficient isolation of such cells thatis cumbersome or impossible with conventional strategies.

Manipulation of T-Cell Populations

In some aspects, this invention provides methods for the manipulation ofT-cells using reversible monomers. In some embodiments, the methodincludes a step of contacting a population of cells expressing a T-cellreceptor with an MHC multimer as described herein under conditionssuitable for the multimer to bind to the T-cell receptor and for a timesufficient for the T-cell receptor/MHC class I interaction to effectTCR-mediated T-cell activation. In some embodiments, the contacting isperformed in vitro. In some embodiments, the contacting is performed exvivo. In some embodiments, the contacting is performed in vivo. In someembodiments, the cells are contacted with an MHC multimer for a timelong enough to activate high-affinity TCR expressing T-cells, but not toactivate low affinity TCR expressing T-cells. In some embodiments, thecells are cells from a subject having an autoimmune disease. In someembodiments, the cells are contacted with an MHC multimer that is loadedwith an antigenic peptide recognized by T-cells that mediate anautoimmune disease. In some embodiments, the method further comprisesmeasuring the quantity of the T-cells targeted by the MHC multimer, forexample, by methods for the identification or detection of T-cellsprovided herein or otherwise known in the art.

Isolated Cell Populations

Some embodiments of this invention provide isolated cells or cellpopulations, for example, isolated native, or non-activated T-cellpopulations obtained by using a reversible multimer or a method asprovided herein. In some embodiments, an isolated cell is provided thathas been contacted with a reversible multimer provided herein andisolated from a cell population based on the cell binding the multimer,for example, by a method for detection and/or isolation described inmore detail elsewhere herein. In some embodiments, the cell is a T-cell.In some embodiments, the T-cell is a native T-cell, or a T-cell that hasnot undergone TCR-mediated cell activation. In some embodiments, thecell has been contacted with an agent releasing the chelate complex bondof the reversible multimer subsequent to its isolation. In someembodiments, the cell is a T-cell recognizing a tumor antigen. In someembodiments, the cell is a T-cell expressing a TCR that binds a tumorantigen with high affinity. In some embodiments, the cell is atherapeutically valuable cell. In some embodiments, the cell is expandedin vitro after isolation, and used in a therapeutic method. In someembodiments, the therapeutic method includes a step of administering thecell to a subject in need thereof, for example, a subject having a tumoror having an elevated risk of developing a tumor expressing a tumorantigen. In some embodiments, a subject at risk of developing a tumorexpressing a tumor antigen is a subject which was diagnosed to have sucha tumor and has undergone surgical removal of the tumor.

Further materials, methods, suitable conditions, and usefulmodifications for the use of reversible MHC multimers as describedherein will be apparent to those of skill in the art. Methods for theuse of conventional multimers can generally be applied to the use of theinventive multimers provided herein with modifications that do notamount to more than routine experimentation. Examples of such methodsare described, for example, in Altman J D, Moss P A, Goulder P J,Barouch D H, McHeyzer-Williams M G, Bell J I, McMichael A J, Davis M M.Phenotypic analysis of antigen-specific T lymphocytes. Science 1996;274: 94-96; Bakker A H, Schumacher T N. MHC multimer technology: currentstatus and future prospects. Curr Opin Immunol 2005; 17: 428-433; Xu XN, Screaton G R. MHC/peptide tetramer-based studies of T cell function.J Immunol Methods 2002; 268: 21-28; Guillaume P, Baumgaertner P, Neff L,Rufer N, Speiser D E, Luescher I F. Novel soluble HLA-A2/Melan-Acomplexes selectively stain a differentiation defective subpopulation ofCD8+ T cells in melanoma patients Int J Cancer 2009; in press; GuillaumeP, Legler D F, Boucheron N, Doucey M A, Cerottini J C, Luescher I F.Soluble major histocompatibility complex-peptide octamers with impairedCD8 binding selectively induce Fasi-dependent apoptosis. J Biol Chem2003; 278: 4500-4509; Neveu B, Echasserieau K, Hill T, Kuus-Reichel K,Houssaint E, Bonneville M, Saulquin X. Impact of CD8-MHC class Iinteraction in detection and sorting efficiencies of antigen-specific Tcells using MHC class I/peptide multimers: contribution of pMHC valency.Int Immunol 2006; 18: 1139-1145; Knabel M, Franz T J, Schiemann M, WulfA, Villmow B, Schmidt B, Bernhard H, Wagner H, Busch D H. Reversible MHCmultimer staining for functional isolation of T-cell populations andeffective adoptive transfer. Nat Med 2002; 8: 631-637; Guillaume P,Baumgaertner P, Angelov G S, Speiser D, Luescher I F.Fluorescence-activated cell sorting and cloning of bona fide CD8+ CTLwith reversible MHC-peptide and antibody Fab′ conjugates. J Immunol2006; 177: 3903-3912; Yao J, Bechter C, Wiesneth M, Härter G, Götz M,Germeroth L, Guillaume P, Hasan F, von Harsdorf S, Mertens T, Michel D,Döher H, Bunjes D, Schmitt M, Schmitt A. Multimer staining ofcytomegalovirus phosphoprotein 65-specific T cells for diagnosis andtherapeutic purposes: a comparative study. Clin Infect Dis 2008; 46:96-105; Chattopadhyay P K, Price D A, Harper T F, Betts M R, Yu J,Gostick E, Perfetto S P, Goepfert P, Koup R A, De Rosa S C, Bruchez M P,Roederer M. Quantum dot semiconductor nanocrystals for immunophenotypingby polychromatic flow cytometry. Nat Med 2006; 12: 972-977; Cebecauer M,Guillaume P, Hozák P, Mark S, Everett H, Schneider P, Luescher, I F.Soluble MHC-peptide complexes induce rapid death of CD8+ CTL. J Immunol2005; 174: 6809-6819; Cebecauer M, Guillaume P, Mark S, Michielin O,Boucheron N, Bezard M, Meyer, B H, Segura J M, Vogel H, Luescher I F.CD8+ cytotoxic T lymphocyte activation by soluble majorhistocompatibility complex-peptide dimers. J Biol Chem 2005; 280:23820-23828; Angelov G S, Guillaume P, Cebecauer M, Bosshard G,Dojcinovic D, Baumgaertner P, Luescher I F. Soluble MHC-peptidecomplexes containing long rigid linkers abolish CTL-mediatedcytotoxicity. J Immunol 2006; 176: 3356-3365; Batard P, Peterson D A,Devêvre E, Guillaume P, Cerottini J C, Rimoldi D, Speiser D E, WintherL, Romero P. Dextramers: new generation of fluorescent MHC classI/peptide multimers for visualization of antigen-specific CD8+ T cells.J Immunol Methods 2006; 310: 136-148; Fahmy T M, Bieler J G, Schneck JP. Probing T cell membrane organization using dimeric MHC-Ig complexes.J Immunol Methods 2002; 268: 93-106; van der Merwe P A, Davis S J.Molecular interactions mediating T cell antigen recognition. Annu RevImmunol 2003; 21: 659-684; Campanelli R, Palermo B, Garbelli S,Mantovani S, Lucchi P, Necker A, Lantelme E, Giachino C. Human CD8co-receptor is strictly involved in MHC-peptide tetramer-TCR binding andT cell activation. Int Immunol 2002; 14: 39-44; Luescher I F, Vivier E,Layer A, Mahiou J, Godeau F, Malissen B, Romero P. CD8 modulation ofT-cell antigen receptor-ligand interactions on living cytotoxic Tlymphocytes. Nature 1995; 373: 353-356; Kao C, Daniels M A, Jameson S C.Loss of CD8 and TCR binding to Class I MHC ligands following T cellactivation. Int Immunol 2005; 17: 1607-1617; Comelli E M, Sutton-SmithM, Yan Q, Amado M, Panico M, Gilmartin T, Whisenant T, Lanigan C M, HeadS R, Goldberg D, Morris H R, Dell A, Paulson J C. Activation of murineCD4+ and CD8+ T lymphocytes leads to dramatic remodeling of N-linkedglycans. J Immunol 2006; 177: 2431-2440; Wooldridge L, Lissina A, Cole DK, van den Berg H A, Price D A, Sewell A K. Tricks with tetramers: howto get the most from multimeric peptide-MHC. Immunology 2009; 126:147-164; Xu X N, Purbhoo M A, Chen N, Mongkolsapaya J, Cox J H, Meier UC, Tafuro S, Dunbar P R, Sewell A K, Hourigan C S, Appay V, Cerundolo V,Burrows S R, McMichael A J, Screaton G R. A novel approach toantigen-specific deletion of CTL with minimal cellular activation usingalpha3 domain mutants of MHC class I/peptide complex. Immunity 2001; 14:591-602; Demotte N, Stroobant V, Courtoy P J, Van Der Smissen P, ColauD, Luescher I F, Hivroz C, Nicaise J, Squifflet J L, Mourad M, GodelaineD, Boon T, van der Bruggen P. Restoring the association of the T cellreceptor with CD8 reverses anergy in human tumor-infiltratinglymphocytes. Immunity 2008; 28: 414-424; Dimopoulos N, Jackson H M,Ebert L, Guillaume P, Luescher I F, Ritter G, Chen W. Combining MHCtetramer and intracellular cytokine staining for CD8(+) T cells toreveal antigenic epitopes naturally presented on tumor cells. J ImmunolMethods 2009; 340: 90-94; Choi E M, Chen J L, Wooldridge L, Salio M,Lissina A, Lissin N, Hermans I F, Silk J D, Mirza F, Palmowski M J,Dunbar P R, Jakobsen B K, Sewell A K, Cerundolo V. High avidityantigen-specific CTL identified by CD8-independent tetramer staining. JImmunol 2003; 171: 5116-5123; Pittet M J, Rubio-Godoy V, Bioley G,Guillaume P, Batard P, Speiser D, Luescher I, Cerottini J C, Romero P,Zippelius A. Alpha 3 domain mutants of peptide/MHC class I multimersallow the selective isolation of high avidity tumor-reactive CD8 Tcells. J Immunol 2003; 171: 1844-1849; Wooldridge L, Scriba T J, MilicicA, Laugel B, Gostick E, Price D A, Phillips R E, Sewell A K.Anti-coreceptor antibodies profoundly affect staining with peptide-MHCclass I and class II tetramers. Eur J Immunol 2006; 36: 1847-1855;Scriba T J, Purbhoo M, Day C L, Robinson N, Fidler S, Fox J, Weber J N,Klenerman P, Sewell A K, Phillips R E. Ultrasensitive detection andphenotyping of CD4+ T cells with optimized HLA class II tetramerstaining. J Immunol 2005; 175: 6334-6343; Day C L, Seth N P, Lucas M,Appel H, Gauthier L, Lauer G M, Robbins G K, Szczepiorkowski Z M, CassonD R, Chung R T, Bell S, Harcourt G, Walker B D, Klenerman P,Wucherpfennig K W. Ex vivo analysis of human memory CD4 T cells specificfor hepatitis C virus using MHC class II tetramers. J Clin Invest 2003;112: 831-842; Mallone R, Nepom G T. MHC Class II tetramers and thepursuit of antigen-specific T cells: define, deviate, delete. ClinImmunol 2004; 110: 232-242; Reijonen H, Kwok W W. Use of HLA class IItetramers in tracking antigen-specific T cells and mapping T-cellepitopes. Methods 2003; 29: 282-288; Vollers S S, Stern L J. Class IImajor histocompatibility complex tetramer staining: progress, problems,and prospects. Immunology 2008; 23: 305-313; Arnold P Y, La Gruta N L,Miller T, Vignali K M, Adams P S, Woodland D L, Vignali D A. Themajority of immunogenic epitopes generate CD4+ T cells that aredependent on MHC class II-bound peptide-flanking residues. J Immunol2002; 169: 739-749; Reche P A, Reinherz E L. Definition of MHCsupertypes through clustering of MHC peptide-binding repertoires.Methods Mol Biol 2007; 409: 163-173; Boniface J J, Rabinowitz J D,Wülfing C, Hampl J, Reich Z, Altman J D, Kantor R M, Beeson C, McConnellH M, Davis M M. Initiation of signal transduction through the T cellreceptor requires the multivalent engagement of peptide/MHC ligands.Immunity 1998; 9: 459-466; Lovitch S B, Unanue E R. Conformationalisomers of a peptide-class II major histocompatibility complex. ImmunolRev 2005; 207: 293-313; Cameron T O, Cochran J R, Yassine-Diab B, SékalyR P, Stern L J. Cutting edge: detection of antigen-specific CD4+ T cellsby HLA-DR1 oligomers is dependent on the T cell activation state. JImmunol 2001; 166: 741-745; Yang J, James E A, Huston L, Danke N A, LiuA W, Kwok W W. Multiplex mapping of CD4 T cell epitopes using class IItetramers. Clin Immunol 2006; 120: 21-32; Blanchet J S, Valmori D, DufauI, Ayyoub M, Nguyen C, Guillaume P, Monsarrat B, Cerottini J C, RomeroP, Gairin J E. A new generation of Melan-A/MART-1 peptides that fulfillboth increased immunogenicity and high resistance to biodegradation:implication for molecular anti-melanoma immunotherapy. J Immunol 2001;167: 5852-5861; Schiavetti F, Thonnard J, Colau D, Boon T, Coulie P G. Ahuman endogenous retroviral sequence encoding an antigen recognized onmelanoma by cytolytic T lymphocytes. Cancer Res 2002; 62: 5510-5516; AliS A, Lynam J, McLean C S, Entwisle C, Loudon P, Rojas J M, McArdle S E,Li G, Mian S, Rees R C. Tumor regression induced by intratumor therapywith a disabled infectious single cycle (DISC) herpes simplex virus(HSV) vector, DISC/HSV/murine granulocyte-macrophage colony-stimulatingfactor, correlates with antigen-specific adaptive immunity. J Immunol2002; 168: 3512-3519; Pardigon N, Darche S, Kelsall B, Bennink J R,Yewdell J W. The TL MHC class Ib molecule has only marginal effects onthe activation, survival and trafficking of mouse small intestinalintraepithelial lymphocytes. Int Immunol 2004; 16: 1305-1313; GuillouxY, Lucas S, Brichard V G, Van Pel A, Viret C, De Plaen E, Brasseur F,Lethe B, Jotereau F, Boon T. A peptide recognized by human cytolytic Tlymphocytes on HLA-A2 melanomas is encoded by an intron sequence of theN-acetylglucosaminyltransferase V gene. J Exp Med 1996; 183: 1173-1183;Rakoff-Nahoum S, Kuebler P J, Heymann J J, E Sheehy M, M Ortiz G, S OggG, Barbour J D, Lenz J, Steinfeld A D, Nixon D F. Detection of Tlymphocytes specific for human endogenous retrovirus K (HERV-K) inpatients with seminoma. AIDS Res Hum Retroviruses 2006; 22: 52-56; LisoA, Colau D, Benmaamar R, De Groot A, Martin W, Benedetti R, Specchia G,Martelli M P, Coulie P, Falini B. Nucleophosmin leukaemic mutantscontain C-terminus peptides that bind HLA class I molecules. Leukemia2008; 22: 424-426; and Matsuki N, Ogasawara K, Takami K, Namba K,Takahashi A, Fukui Y, Sasazuki T, Iwabuchi K, Good R A, Onoe K.Prevention of infection of influenza virus in DQ6 mice, a human model,by a peptide vaccine prepared according to the cassette theory. Vaccine1999; 17: 1161-1168. All references are incorporated herein in theirentirety by reference for disclosure of methods and materials useful forthe generation, isolation, and or purification of MHC multimers and forstaining, detection, and/or isolation of cells using MHC multimers.

The function and advantage of these and other embodiments of the presentinvention will be more fully understood from the examples below. Thefollowing examples are intended to illustrate the benefits of thepresent invention, but do not exemplify the full scope of the invention.

EXAMPLES Example 1

Some aspects of this invention provide a novel type of MHC multimers inwhich MHC class I-peptide monomers are conjugated to phycobilins (PE orAPC) or quantum dots (Qdots) via chelate complexes of histidine (His)tags and Ni²⁺-nitrilotriacetic acid (NTA). Hexa (His₆), dodeca (His₁₂)or tandem hexa (2×His₆) histidine tags were fused to HLA-A*0201 (A2)heavy chain and A2-peptide monomers obtained in good yields byrefolding. Mono, di and tetra NTA derivatives were synthesized and theirinteractions with His tagged monomers studied by surface plasmonresonance (SPR) and by CD8+ T cell staining experiments. The resultsdescribed here indicate that the affinity (K_(D)) increases in the orderHis₆>His₁₂>2×His₆ and mono>di>tetra NTA, respectively, spanning severalorders of magnitudes. Staining experiments on influenza-specific CD8+ Tcell clones and populations with NTA-His tag A2/Flu₅₈₋₆₆ multimersindicated that: i) multimers containing 2×His₆ tagged complexes andshort di- or tetra NTA moieties were equal or superior compared toconventional multimers; ii) di-NTA or tetra-NTA can be directly coupledto the phycobilin proteins or Qdots, which circumvents the use of biotinand streptavidin, and renders synthesis simpler and cheaper; iii) thesereagents are molecularly better defined than conventional multimers andhence allow better analysis of binding data; iv) NTA-His tag builtmultimers dissociated rapidly in the presence of 100 mM imidazol(t_(1/2)<1 min), which allows sorting of bona fide antigen-specific CD8+T cells without inducing activation dependent cell death.

MATERIALS AND METHODS

Abbreviations used herein include: APC: allophycocyanine; β2m:beta-2-microglobulin; BSP: biotinylation sequence peptide; DIEA:di-isopropyl-ethyl-amine; EDIC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; GFC: gel filtration chromatography; Flu: influenza matrix;HOBt: 1-hydroxy-benzo-triazole; NHS: N-hydroxysuccinimide; NTA:nitrilotriacetic acid; PE: phycoerythrin; Qdot: quantum dot; SPR:surface plasmon resonance; TBTU:O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate.

Chemical Synthesis

Protected amino acids and 2-chlorotrityl resin were obtained fromReactolab (Servion, Switzerland), TBTU and HOBt were from Multisynthec(Witten, Germany), maleimide-NTA from Dojindo Laboratories (Kumamoto,Japan). RP-HPLC analyses were performed on Waters HPLC stationconsisting of two 515 pumps and a Waters 996 photodiode array detector.The purity of all peptides was examined by analytical HPLC on a C18reverse phase column (Uptisphere 5 μm C₁₈ particles, 250×4.6 mm) andwhich was eluted with a linear gradient rising from 100% of 0.1% TFA inH₂O to 50% of 0.08% TFA in CH₃CN in 30 min at a flow rate of 1 ml/min.peptides were purified on a semi preparative column (Kromasil 15 μm C₁₈particles, 250×20 mm) at a flow rate of 3 mL/min, with UV monitoring at214 nm. The purified peptides were characterized for correct Mr usingmatrix-assisted laser desorption ionization time-of-flight massspectrometer (Micromass QTOF Ultima) (Waters Ltd, En Yvelines Cedex,France).

Synthesis of Linear Peptides

Synthesis of linear peptides was carried out manually in a syringefitted with a sintered frit using Fmoc/tBu strategy. Coupling reactionswere performed using 2 equiv of N-α-Fmoc-protected amino acid relativeto the resin loading, activated in situ with 2 equiv of TBTU, 2 equiv ofHOBt and 4 equiv of DIEA in DMF (10 mL/g resin) for 1 h. Couplingcompletion was verified by Kaiser tests. N-α-Fmoc protecting groups wereremoved by treatment with a piperidine/DMF solution (1:4) (10 mL/gresin) for 5 min. The process was repeated twice, and the completion ofdeprotection was checked by the UV absorption of the piperidine washingat 299 nm. Peptides were obtained by cleavage of the resin withTFA/H₂O/TIPS (92.5/2.5/5) for 3 h and after filtering of the resinprecipitated by addition of ether, filtered off, dissolved in water,purified by semi-preparative HPLC and lyophilized.

Coupling of Maleimide-NTA to Linear Peptides

Linear free SH containing peptides were dissolved in phosphate buffer(0.1M, pH 7.2) at a concentration of 0.1 M. Two equivalents of Mal-NTA(relative to SH) were added and the mixture stirred for 1 h under argon.The product was further purified by semi-preparative HPLC and analyzedby Electrospray ionisation on a Micromass QTOF Ultima instrument.Alternatively, in the strategy shown in FIG. 13, the backbone peptidewas reacted via in situ carboxyl activation with N^(α),N^(α)-Bis[(tert-butyloxycarbonyl)methyl]-L-lysine tert-butyl ester(H₂N-NTA(tBu)₃), which was synthesized as follow: tert-butylbromoacetate (1.59 ml, 10.8 mmol) and DIEA (2.30 ml, 13.5 mmol) wereadded sequentially to a solution of NE-benzyloxycarbonyl-lysinetert-butyl ester (1.00 g, 2.7 mmol) in DMF (25 ml). The reaction vesselwas purged with N₂ and then continuously stirred overnight at 55° C. Thevolatiles were evaporated in vacuo at 60° C. Cyclohexane/ethylacetate(3:1, 15 ml) solution was added to the partially solidified reactionmixture. The resulting slurry was filtered over sintered glass funneland the precipitate washed three times with the same solvent (3×10 ml).The filtrate was concentrated under reduced pressure and the resultingyellow powder dissolved in methanol (50 ml), the solution purged with N₂followed by addition of 10% Pd/C (20 mg). The reaction mixture wasvigorously stirred for 6 hours under H₂ atmosphere at room temperature.Pd/C was removed by filtration over celite and the volatiles removedunder reduced pressure. The product was purified by silicachromatography with chloroform/methanol (3:1) as the eluent. Yield: 1.03g (2.4 mmol; 91%). The linear peptideH-K(aminocaproyl-biotin)-PEG-A-E*-A-E*-OH (*: Fmoc-E-OtBu) wassynthesized on an ABI433 peptide synthesizer. Double coupling of eachFmoc-protected amino acid were performed using DIPC and HOBt as couplingreagent. Fmoc was removed by 3×5 min treatment with piperidine 20% inDMF. Each cycle was followed by an acetylation (N-capping) to preventthe synthesis of truncated peptides. Final cleavage was performed inTFA/TIPS/H₂O (92.5/5/2.5) for 2 h. The peptide was precipitated withcold ether, dissolved in water and purified by semi-preparative HPLC.Similarly, the synthesis of SH-NTA2 was performed assembling the linearC(Acm)-PEG-C(Trt)-G-C(Trt) on a chlorotrityl resin. After TFA treatmentthe linear peptide C(Acm)-PEG-C(SH)-G-C(SH) was coupled to maleimide-NTAas described previously. 1 μmol of lyophilized peptide was dissolved in100 μl of AcOH 20%, the pH was adjusted to 4 with aqueous ammonia. 3μmol of mercury(II) acetate were added and the mixture stirred for 1 h.5 μmol of DTT were then added and the mixture stirred for 1 additionalhour. The desired product was directly purified by semi-preparative HPLCand analyzed by mass spectrometry.

Surface Plasmon Resonance Experiments

Affinity measurements were performed on a Biacore 3000 instrumentequipped with SA coated chips. The eluent buffer (10 mM HEPES, 150 mMNaCl, 50 mM EDTA at pH 7.4) and the dispenser buffer (10 mM HEPES, 150mM NaCl, 0.005% polysorbate 20, 3 mM EDTA) were filtered and degassedprior to use. Two or five-fold dilutions of His-tagged A2/Flu₅₈₋₆₆monomers were freshly prepared in eluent buffer before each experiment.Loading of biotin containing NTA peptides was performed in freshsolution of NTA-biotin peptides dissolved in eluent buffer. A RU(resonance unit) of 100 was used in all experiment. Loading of NTA withNi²⁺ was performed by injecting NiCl2 solution (500 mM in eluent buffer)and regeneration of the chip with imidazole (500 mM in water) followedby a regeneration solution (10 mM HEPES, 150 mM NaCl, 0.005% polysorbate20, 350 mM EDTA at pH 7.4). All binding experiments were performed at aflow rate of 100 μl/min, starting with a 1 min injection of NiCl₂solution. Each sample was injected for 5 min followed by 5 min ofundisturbed dissociation time. The regeneration procedure consisted oftwo subsequent 1-min injections of imidazole and regeneration solution.

HLA-A2 Flu 58-66 multimers

HLA-A*0201-Peptide Complexes

HLA-A*0201 (A2) heavy chains containing the His tags shown in FIG. 2Aexpressed as inclusion bodies in E. Coli as described previously for theBSP containing heavy chain (3). The different heavy chains were refoldedin the presence of hβ2m and the influenza matrix peptide₅₈₋₆₆(GILGFVFTL, SEQ ID NO: 12) and purified on a Superdex S75 column asdescribed (3).

Preparation of A2/Flu 58-66 Multimers

Conventional streptavidin PE multimers were prepared as described (3).NTA-streptavidin PE multimers were prepared in two steps. First,streptavidin PE conjugate (Invitrogen) was incubated with NTA-biotinpeptides (five-fold molar excess) at 4° C. for 1 h followed byincubation for 30 min with NiSO4 (10 mM). Excess of reagents wereremoved by means of min-spin columns (Zeba™ Spin Desalting Columns(Thermo scientific). His tagged monomeric A2/Flu complexes were mixedwith Ni-NTA-biotin-streptavidin PE conjugate at a ten-fold molar excessand kept at 4° C. until use. NTA₂-PE conjugates were prepared by firstreacting PE (Sigma) (50 nM) in 0.1 M phosphate buffer, pH 7.2 with 10 mM(or as indicated) SM(PEG)₂ (Pierce) at room temperature for 2 h. Excessreagents were removed by centrifugation through spin columns (supplier).The resulting PE-maleimide conjugates were incubated under argon in 100mM phosphate buffer, pH 7.0 with 50 mM cysteine-di-NTA at roomtemperature for 1-2 h (FIG. 3E). After incubation for 30 min with NiSO4(10 mM), excess reagents were removed by centrifugation through spincolumns and the concentration of the resulting Ni²⁺NTA₂-PE wasdetermined by Bradford.

Cells, Staining Procedures and Flow Cytometry

Cells Under Study

The HLA-A*0201-restricted, influenza matrix peptide₅₈₋₆₆-specific CD8+ Tcell clones were obtained by limiting dilution cloning from bulkcultures. CD8+ PBMC from healthy donors were prepared by negativeselection and were stimulated with Flu₅₈₋₆₆ peptides as described (4).The clones were re-stimulated in 24-well plates every 15 d in RPMI 1640medium supplemented with 8% human serum, rIL-2 (150 U/ml) (Hoffmann-1aRoche Ltd, Basel, Switzerland) with PHA (1 μg/ml; Sodiag S A, Losone,Switzerland) and 1×10⁶/ml irradiated allogeneic PBMC (3000 rad) asfeeder cells. Bulk cultures were prepared by one or two peptidestimulations of CD8+ T cells obtained from PBMC from a DR4+ healthydonor (IFL).

Multimer Binding Assays, Flow Cytometry and Analysis.

For binding studies CD8+ T cells (5×10⁴) were incubated for 30-45 min atambient temperature with graded concentrations of the differentA2/Flu₅₈₋₆₆ complexes in 20 μl of FACS buffer (OptiMEM (Invitrogen AG,Basel, Switzerland) supplemented with 0.5% BSA (Sigma-Aldrich), 15 mMHEPES, 5 mM EDTA, and 5 mM NaN₃). In some experiments cells wereincubated an additional 20 min at 4° C. with anti-CD8-FITC (ImmunoTools). After 30-fold dilution in FACS buffer, cell-associatedfluorescence was measured on a LSR11 flow cytometer (BD Biosciences).Background binding was determined on a A2/Mealn-A₂₆₋₃₅(ELAGIGILTV, SEQID NO: 13) clone (EM28-9.24) and was subtracted from the cognatestaining. Data were processed using the FlowJo software (Tree Star, Inc.Ashland, Oreg.). For dissociation experiments CD8+ T cells wereincubated for 45 min at 4° C. with 10 nM of multimers in FACS buffer,diluted 200× fold in FACS buffer and after various periods of incubationat 4° C., cell-associated fluorescence was determined by flow cytometry(0 to 60 min). In some dissociation experiments with NTA multimers FACSbuffer was supplemented with imidazol hydrochloride (50 or 100 mM).

Results and Discussion

To build sufficiently stable MHC-peptide multimers on Ni²⁺NTA-His tagchelate complexes, we examined the interaction of different His tags andNTA moieties. In the minimal subunit complex one NTA forms a coordinatecomplex with a Ni²⁺ ion, which in turn can bind two imidazoles, i.e.side chains of histidines (FIG. 1A). Since this subunit complex is notsufficiently stable, we prepared HLA-A*02010/Flu₅₈₋₆₆ monomerscontaining C-terminal a hexa-histidine (His₆), a dodeca-histidine(His₁₂) or double hexa-histidine tag (2×His₆) (FIG. 2A). On the otherhand we synthesized mono, di- and tetra-NTA compounds, which containedbiotin (FIGS. 3A-C). Because the binding of biotin to streptavidin isexceedingly strong (K_(D) ˜10⁵ M), we used streptavidin eitherconjugated to PE or immobilized on SPR sensor chips to stably bind thedifferent biotinylated NTA derivatives. Addition of His tagged A2/Flumonomers to PE-streptavidin yielded multimers in which MHC-peptidecomplexes are conjugated via the NTA-His complexes (FIG. 1C). On theother hand this strategy allowed accurate SPR measurements of thedifferent NTA-His tag interactions (FIG. 3A).

Identification of Suitable His Tags and NTA Linkers

To identify a suitable His tag we prepared A2 heavy chains containingC-terminally added His₆, His₁₂ and 2×His₆ (tandem) His tags (FIG. 2A).These heavy chains were refolded with 132m and Flu matrix₅₈₋₆₆ peptidefollowing established procedures (3). The refolding efficiency of the2×His₆ tagged complex was nearly as high (98%) as the one of the BSPcomplex (FIG. 2B). For the His₆ tagged complex the efficiency wasapproximately 85% and for the His₁₂ tagged one only 60%. Similar resultswere obtained when using other peptides (e.g. Melan-A₂₆₋₃₅ orNY-ESO-1₁₅₇₋₁₆₅).

We then synthesized the NTA linker shown in FIGS. 3A-C and examinedtheir binding of the His tagged A2/Flu₅₈₋₆₆ monomers by SPR. To this endstreptavidin coated sensor chips were loaded with the biotinylated NTAcompounds and the binding of the monomers measured by the changes inresonance units (RU). On mono-NTA (FIG. 3A) coated chips, thedissociation constant (K_(D)) decreased dramatically from the His₆, tothe His₁₂ and 2×His₆ tagged complexes (from 4100 to 34 nM) (FIG. 4B). OnNTA₂ (FIG. 3B) coated chips the K_(D) values were lower still, reaching12 nM for the 2×His₆ tagged A2/Flu complex. This increase in affinitywas largely accounted for by decreased dissociation rates, i.e. thechelate complexes become increasingly more stable. These findings areconsistent with reports showing that the affinity of His tags for Ni²⁺NTA moieties dramatically increases with their valence and that NTA₃compounds bind His₆ tags with sub-nanomolar K_(D) (13-15).

We next examined staining of the Flu matrix-specific clone 81P1 byA2/Flu₅₈₋₆₆ multimers containing the same His tags and streptavidin-PEsaturated with biotin-Ni²⁺ NTA moieties. The 20° C. binding isotherms ofthe multimers containing NTA₂ were consistently higher than thoseobtained of mono NTA containing multimers (FIG. 5A). In both casesbinding was strongest with multimers containing the 2×His₆ tag.Multimers containing this His tag and NTA₂ containing exhibited higherbinding than conventional multimers. While the binding values for NTA₂complexes with His₁₂ containing complexes was only slightly lower, allother combinations exhibited substantially weaker binding and hence werenot further investigated. Similar results were obtained on other Fluclones (data not shown).

To compare the binding of A2/Flu multimers containing NTA₄ and NTA₂, weperformed similar binding experiments on cloned BCB 70 cells. The 20° C.binding isotherms showed that NTA₄ multimers bound more avidly than NTA₂or conventional multimers (FIG. 5B). While in the case of NTA₄ thebinding of multimers containing the His₁₂ or 2×His₆ tag was essentiallythe same, for the NTA₂ multimers those containing the 2×His₆ tagexhibited better binding compared to those containing the His₁₂ tag.

It should be noted that staining results critically depend on theconfiguration of the NTA molecule. For example, we initially synthesizedanother biotin-NTA₂ compound, in which lysine NTA was coupled via amidebonds to a linear peptide containing two orthogonal carboxyl side chainsabout 14 Å apart (FIG. 13). Because this is longer (by about 4.4 Å)compared to the other biotin-NTA₂ (FIG. 3B, FIG. 12), we refer to thislinker as short and the other as long. Binding isotherms on cloned 81P1cells at 20° C. cells indicated that A2/Flu multimers containing theshort performed better than the multimers containing the long NTA₂moiety (FIG. 15). While multimers containing the short NTA₂-biotin and2×His₆ tagged A2/Flu complexes exhibited superior binding thanconventional multimers (FIG. 15A), all multimers containing the longdi-NTA showed inferior binding (FIG. 15B). Moreover, the bindinghierarchy of multimers containing the differently tagged A2/Flucomplexes and the short, respectively the long NTA₂-biotin wereremarkably disparate. We also tested multimers containing the commercialNTA-biotin (FIG. 3F) and observed substantially lower binding comparedto multimers containing the long NTA-biotin (FIG. 3A)(data not shown).

Taken collectively, these results demonstrate that MHC class I-peptidemultimers can be built on NTA-His tag chelate complexes that performequal or better than conventional BSP multimers. The affinity andstability of Ni²⁺NTA-His tag complexes depends not only on the number ofNi²⁺NTA entities and histidines, but also on their configuration. Thisis primarily explained by the number of subunit Ni²⁺NTA-histidinechelate complexes that de facto can be formed (FIG. 1A). Our SPR bindingstudies indicated that complexes containing 2×His₆ tagged A2/Flucomplexes have higher affinities (i.e. lower K_(D) values) and slowerdissociation kinetics (i.e. lower k_(off) values (FIG. 4B). This isconsistent with previous reports and most likely explained by that twoHis₆ tags joined by a flexible spacer can interact with more Ni²⁺NTAentities than the relative rigid His₁₂ tag 0. Our multimer bindingstudies on cells are consistent with this, although in the case of NTA₄containing multimer differences were marginal (FIG. 5B). On the otherhand our staining results indicate that Ni²⁺NTA entities form morestable complexes with His tags when they have long flexible side chains,yet short intervening linkers (FIGS. 12-15). Little is known on howbinding parameters of NTA-His tag interactions depend on the spatialconfiguration of the NTA moiety. One study showed that tri-NTA compoundsmost avidly bind to His6 tags when they contain minimal spacers (15). Tobetter understand this relationship, we are currently testing additionaldi and tetra NTA molecules.

We performed staining experiments with multimers containing streptavidinQdots₆₀₅ loaded with biotin-NTA₂ on 81P1 cells. As shown in FIG. 16 thestaining of was similar for all monomers tested, i.e. the nature of theHis tag had little effect on the staining. These Qdots are larger thanPE, contain more streptavidin on their surface and therefore more NTA₂groups; as discussed below, the density of NTA groups on a surface isanother factor determining the stability of complexes with His taggedproteins.

NTA-his Tag Built Multimers are Reversible

We next assessed the dissociation kinetics of A2/Flu multimers built onNTA₂-biotin-PE streptavidin in the presence of different concentrationsof imidazol and/or EDTA. Cloned 81P1 cells were stained in the cold withmultimers containing NTA₂ and His₁₂ or 2×His₆ tags, washed and incubatedfor different periods of time at 4° C. in media containing imidazol. Thestaining of 2×His₆ tag containing multimers decreased more rapidly thanthe staining of His₁₂ tag containing multimers (FIGS. 6A, B). In thepresence of 50 mM imidazole half maximal dissociation was observed after8.1 and 54 min, respectively. In the presence of 50 mM imdidazol plus 20mM EDTA the dissociation rate increased and half maximal dissociationwas reached after 12.1 and 6.6 min, respectively. In the presence ofEDTA alone dissociations were very slow in both cases (data not shown).By contrast, rapid dissociations were observed in the presence of 100 mMimidazole, with half maximal dissociations after 1.3 and 0.7 min,respectively. At higher concentrations of imidazole the dissociation wasfurther accelerated, but in some cases cell viability was affected.

Analogous dissociation experiments were performed on cloned BCB 70 cellswith multimers containing NTA₂- and NTA₄ biotin-streptavidinA2/Flu₅₈₋₆₆multimers. For NTA₂ multimers similar results were obtainedas in the previous experiment (FIGS. 6A and 7A). The dissociations forNTA₄ multimers were slower. In the presence of 50 mM imidazol thehalf-life for the His₁₂ containing multimer was in the range of hoursand for the 2×His6 containing one about 25 min (FIG. 7B). However, inthe presence of 100 mM imidazol dissociations were much faster, withhalf-lives of 2 and 1.2 min for the His₁₂ and 2×His₆ containingmultimers, respectively. It is interesting to note that while thephysical dissociation of 2×His₆ tagged molecules from Ni²⁺ NTA wasslower compared to His₁₂ tagged molecules (FIG. 4B), the inverse wastrue when the complexes were dissociated by addition of free imidazol(FIGS. 6, 7).

Preparation, Validation and Application of Biotin Free NTA₂-PE MHC ClassI-Peptide Multimers.

Based on the finding that multimers containing 2×His₆ tagged MHC classI-peptide monomers and streptavidin-PE saturated with biotinylated shortNTA₂ were sufficiently stable to efficiently stain CD8+ CTL (FIGS. 1C,2-5), we coupled NTA₂ directly to PE and produced multimers by loadingthese with 2×His₆ tagged MHC class I-peptide monomers. To this end PE,which has 24 surface exposed lysine residues, was reacted first with thewater soluble maleimide-hydroxy-succinimide ester SM(PEG)₂. Theresulting maleimide conjugated PE was subsequently reacted with theNTA₂-cysteine (see FIG. 3E), yielding stable PE-NTA₂ conjugates bythioether formation. (FIG. 8).

To find out what degree of conjugation of PE with NTA₂ was needed toobtain efficient NTA₂-PE multimer staining, we reacted PE with differentconcentration of SM(PEG)₂. The resulting PE maleimide derivatives wereexhaustively alkylated with NTA₂-cysteine (FIG. 3E) and the resultingNTA₂-PE conjugates loaded with A2/Flu monomers carrying different Histags. The efficiency of all NTA₂-PE multimers to stain cloned 81P1 CTLincreased with the density of NTA₂ groups on PE (FIG. 17). While at lowdegrees of conjugation the multimers containing the 2×His₆ tag exhibitedsuperior binding, at high degrees the His₁₂ tag containing multimerperformed equally well and even the His₆ containing multimer lagged onlylittle behind. This is reminiscent to our experiments with Qdots (FIG.16), but different from the multimer staining withNTA-biotin-streptavidin multimers in which those containing the 2×His₆tag performed clearly better than those containing the His₁₂ tag or asimple His₆ tag (FIG. 5A). We argue that at high densities of NTA groupsHis tags can cooperatively interact with adjacent NTA moieties, whereasat low densities the binding strengths relies primarily on theinteractions between individual His tags and NTA moieties.

We next performed binding isotherms on cloned 81P1 cells comparingconventional A2/Flu BSP multimers with multimers containing NTA₂-PE andHis₁₂ or 2×His₆ tagged monomers. At all temperatures tested, both NTA₂multimers exhibited a stable binding plateau above 4 nM multimerconcentration. By contrast the binding of the BSP multimer increasedover the whole concentration range tested (FIG. 9). This is explained bythat NTA multimers are molecularly better defined than BSP multimers.While the NTA multimers consist of one PE conjugated with variablenumbers of A2/Flu monomers, conventional multimers contain multiplecomplexes of different sizes and stoichiometries. This heterogeneitystems from the conjugation of PE with streptavidin (2, 3) and thereforethe NTA-biotin-streptavidin PE multimers are equally heterogeneous, asreflected by their binding isotherms (FIG. 5, FIG. 18). Importantly,because NTA multimers are better defined, they allow more conclusivebinding analysis (e.g. Scatchard analysis) than conventional BSPmultimers, i.e. can provide more information on given antigen-specific Tcells.

So far all binding experiments were performed on Flu-specific CTLclones. Due to clonal variations such data may not be generallyrepresentative. We therefore repeated the binding studies on apopulation of Flu-specific CD8+ T cells derived from peptide stimulatedPBMC of a DR4+ healthy donor. As shown in FIG. 18, the 20° C. bindingisotherms on this polyclonal population exhibited essentially the samepicture as the one obtained on clones, namely: i) the binding of NTA₄multimer binding was higher than of NTA₂ multimer; ii) multimerscontaining the 2×His₆ tag bound better than those containing the His₁₂tag in the case of NTA₂, but not NTA₄ reagents; iii) the NTA₂-PEmultimer exhibited clear saturation, whereas all streptavidin-PEcontaining ones did not; and iv) the NTA₄ and NTA₂-PE multimersexhibited the highest avidity, i.e. lowest concentrations for halfmaximal binding.

Finally we performed dissociation kinetics on cloned BCB 70 cells formultimers containing NTA₂-PE and 2×His₆ tagged A2/Flu complexes. In thepresence of 50 mM imidazol half maximal dissociation was reached at 4°C. after about 12 min, at 20° C. after 1.8 min and at 37° C. after 1 min(FIG. 10). In the presence of 100 mM imidazol the dissociations werevery rapid with half-lives below 1 min; especially at 20° C. and 37° C.the dissociations were too fast to be measured accurately by this flowcytometric analysis.

Reversible MHC Class I-Peptide Complexes Allow Sorting ofAntigen-Specific CD8+ T Cells without Inducing Activation Dependent CellDeath.

In previous studies we have shown that MHC-1-peptide multimers induceextensive activation-induced death of CD8+ CTL and that this seriouslycompromises multimer sorting or cloning of antigen-specific CD8+ T cells(5, 6). To circumvent this, we previously made DTB (des-thio-biotin), alow affinity biotin variant, multimers which dissociate in the presenceof free biotin (5). We previously demonstrated that antigen-specificCD8+ T cells cloned or sorted with DTB multimers are superior comparedto cells sorted/cloned with conventional BSP multimers in terms of cellviability and functionality. To find out whether NTA built multimerswould offer the same advantage, we first compared the dissociationkinetics of A2/Flu DTB multimers, with multimers containing NTA₂-PE and2×His₆ tagged A2/Flu complexes. As shown in FIG. 19 DTB-streptavidinA2/Flu₅₈₋₆₆ multimers in the presence of 2 mM biotin dissociatedconsiderably slower than NTA₂-PE-2×His₆ multimers in the presence of 100mM imidazole. The differences were especially striking at 4° C. and 20°C.

To directly examine the usefulness of A2/Flu NTA₂-PE 2×His₆ multimersfor FACS sorting, cloned BCB 70 cells were stained with this orconventional BSP multimers. FACS sorted cells were washed once with 100mM imidazol and viable cells enumerated 1, 2 or 3 d afterwards. As shownin FIG. 11A the percentage of viable cells of NTA₂-PE multimer sortedcells was slightly lower compared to untreated cells or cells washedonce with cold imidazol. By contrast cells sorted with conventionalmultimers exhibited only 42% viable cells after 1 d and merely 20% 3dafter sorting. Although these experiments need to be extend by includingsorting of polyclonal populations and by functional analysis, ourresults strongly argue that NTA₂-PE 2×His₆ multimers are equivalent orsuperior to reversible DTB multimers; the beneficial effects of this wehave described in detail in a previous study (5). The same conjugationstrategy can be equally applied to prepare MHC class II multimers orantibody conjugates, i.e. is universally applicable for the preparationof reversible protein conjugates.

In sum, our results demonstrate that MHC class I-peptide multimers canbe prepared based on NTA-His tag chelate complexes that perform equallywell or better than conventional BSP multimers. Importantly, because NTAcomplexes rapidly dissociate in the presence of imidazol, they allowisolation of bona fide antigen-specific CD8+ T cells by FACS or MACS,which is considerably advantage to BSP multimers. Because NTA builtmultimers contain neither biotin nor streptavidin, they are simpler andcheaper to prepare.

REFERENCES

-   1. Altman J D, Davis M M. MHC-peptide tetramers to visualize    antigen-specific T cells. Curr Protoc Immunol. 2003; Chapter 17:Unit    17.3.-   2. Guillaume P, Dojcinovic D, Luescher I F. Soluble MHC-peptide    complexes: tools for the monitoring of T cell responses in clinical    trials and basic research. Cancer Immun. 2009; 9:7.-   3. Guillaume P, Legler D F, Boucheron N, Doucey M A, Cerottini J C,    Luescher I F. Soluble major histocompatibility complex-peptide    octamers with impaired CD8 binding selectively induce Fas-dependent    apoptosis. J Biol. Chem. 2003; 278:4500-9.-   4. Guillaume P, Baumgaertner P, Neff L, Rufer N, Wettstein P,    Speiser D E, Luescher I F. 2010. Novel soluble HLA-A2/MELAN-A    complexes selectively stain a differentiation defective    subpopulation of CD8+ T cells in patients with melanoma. Int J    Cancer 127:910-23.-   5. Guillaume P, Baumgaertner P, Angelov G S, Speiser D, Luescher    I F. Fluorescence-activated cell sorting and cloning of bona fide    CD8+ CTL with reversible MHC-peptide and antibody Fab′    conjugates. J. Immunol. 2006; 177:3903-3912.-   6. Cebecauer M, Guillaume P, Hozák P, Mark S, Everett H, Schneider    P, Luescher I F. Soluble MHC-peptide complexes induce rapid death of    CD8+ CTL. J. Immunol. 2005; 174:6809-19.-   7. Knabel M, Franz T J, Schiemann M, Wulf A, Villmow B, Schmidt B,    Bernhard H, Wagner H, Busch D H. Reversible MHC multimer staining    for functional isolation of T-cell populations and effective    adoptive transfer. Nat. Med. 2002; 8:631-7.-   8. Neudorfer J, Schmidt B, Huster K M, Anderl F, Schiemann M,    Holzapfel G, Schmidt T, Germeroth L, Wagner H, Peschel C, Busch D H,    Bernhard H. Reversible HLA multimers (Streptamers) for the isolation    of human cytotoxic T lymphocytes functionally active against tumor-    and virus-derived antigens. J Immunol Methods. 2007; 320:119-131.-   9. Knecht S, Ricklin D, Eberle A N, Ernst B. Oligohis-tags:    mechanisms of binding to Ni2+-NTA surfaces. J Mol. Recognit. 2009;    22:270-9.-   10. Cao H, Lin R. Quantitative evaluation of His-tag purification    and immunoprecipitation of tristetraprolin and its mutant proteins    from transfected human cells. Biotechnol Prog. 2009; 25:461-7.-   11. Khan F, He M, Taussig M J. Double-hexahistidine tag with    high-affinity binding for protein immobilization, purification, and    detection on ni-nitrilotriacetic acid surfaces. Anal Chem. 2006;    78:3072-9.-   12. Steinhauer C, Wingren C, Khan F, He M, Taussig M J, Borrebaeck    C A. Improved affinity coupling for antibody microarrays:    engineering of double-(His)₆-tagged single framework recombinant    antibody fragments. Proteomics. 2006 6:4227-34.-   13. Lata S, Reichel A, Brock R, Tampé R, Piehler J. High-affinity    adaptors for switchable recognition of histidine-tagged proteins. J    Am Chem. Soc. 2005; 127:10205-10215.-   14. Huang Z, Park J I, Watson D S, Hwang P, Szoka F C Jr. Facile    synthesis of multivalent nitrilotriacetic acid (NTA) and NTA    conjugates for analytical and drug delivery applications. Bioconjug    Chem. 2006; 17:1592-600.-   15. Huang Z, Hwang P, Watson D S, Cao L, Szoka F C.    Tris-Nitrilotriacetic Acids of Subnanomolar Affinity Toward    Hexahistidine Tagged Molecules. Bioconjug Chem. 2009 Aug. 3.

All references listed are incorporated herein in their entirety byreference.

Example 2

The methods and materials described herein are universally applicable togenerate reversible MHC multimers by conjugating a plurality of MHCmolecules to a multivalent carrier molecule via a chelate complex bond.While the invention is not limited to specific MHC molecules, Tables 1and 2 provide exemplary MHC molecules and exemplary antigenic peptidesthat can be used to produce empty or peptide-loaded reversible MHCmolecules using the concepts, methods, and materials provided by aspectsof this invention and described in more detail elsewhere herein. FurtherMHC molecules and antigenic peptides are known in the art and described,for example, in the Tetramer Collection of the Ludwig Institute forCancer Research (see Guillaume P, Dojcinovic D, Luescher I F. LICRtetramer collection: Soluble tetrameric MHC/peptide complexes toidentify and monitor tumor antigen-specific T cells. Cancer Immun 2009;URL: http://www.cancerimmunity.org/tetramers/; both the publication andthe online database are incorporated herein by reference in theirentirety for disclosure of useful MHC molecules and antigenic peptidesaccording to aspects of this invention).

TABLE 1Exemplary MHC class I molecules and antigenic peptides useful for thegeneration of reversible multimers. SEQ ID MHC Protein Position PeptideNO HLA- EBV BMLF1 259-267 GLCTLVAML 14 A*0201 hCMV pp65 495-503NLVPMVATV 15 HIV RT 896-904 ILKEPVHGV 16 Influenza M1 58-66 GILGFVFTL 17MAGE-10 254-262 GLYDGMEHL 18 MELAN-A/ 26-35 ELAGIGILTV 19 MART-1NY-ESO-1 157-165 SLLMWITQC 20 SLLMWITQA 21 Tyrosinase 369-377 YMNGTMSQV22 HLA- Influenza NP 44-52 CTELKLSDY 23 A*0101 MAGE-3 168-176 EVDPIGHLY24 Tyrosinase 146-156 SSDYVIPIGTY 25 243-251 KCDICTDEY 26 HLA- AFP158-166 FMNKFIYEI 27 A*0201 325-334 GLSPNLNRFL 28 BK 90-99 STARIPLPNL 29polyomavirus 108-116 LLMWEAVTV 30 VP1 B-Raf 597-606 LLTEKSRWSV 31 CAMEL 1-11 MLMAQEALAFL 32 CEA 605-613 YLSGANLNL 33 YLSGADLNL 34 YLSGANLDL 35691-699 IMIGVLVGV 36 694-702 GVLVGVALI 37 GLLVGVALI 38 GVLVGVALV 39GLLVGVALV 40 CSPG4/HMW- 561-569 SLMVILEHT 41 MAA 769-777 ILSNLSFPV 421063-1071 LLFGSIVAV 43 2238-2246 LILPLLFYL 44 cytokeratin-18 365-373ALLNIKVKL 45 DCT/TRP-2 180-188 SVYDFFVWL 46 EBV NA-6 284-293 LLDFVRFMGV47 EBV BMLF1 259-267 GLATLVAML 48 Fibromodulin 250-259 YMEHNNVYTV 49G250/CA-IX 24-32 QLLLSLLLL 50 254-262 HLSTAFARV 51 gp100/Pmel 17 154-162KTWGQYWQV 52 209-217 ITDQVPFSV 53 IMDQVPFSV 54 280-288 YLEPGPVTA 55YLEPGPVIA 56 457-466 LLDGTATLRL 57 476-485 VLYRYGSFSV 58 hCMV pp65 14-22VLGPISGHV 59 hepatitis capsid 18-27 FLPSDFFPSV 60 protein HER2/neu369-377 KIFGSLAFL 61 HERV 69-77 DLNNFCQKV 62 HERV-K-Mel MLAVISCAV 63HPV16 E7 11-20 YMLDLQPETT 64 86-93 TLGIVCPI 65 influenza NA 213-221CVNGSCFTV 66 CVNGSCFTI 67 JC polyomavirus 36-44 SITEVECFL 68 VP1 LAGE-186-94 RLLQLHITM 69 103-111 ELVRRILSR 70 MAGE-3 112-120 KVAELVHFL 71157-166 SLQLVFGIEL 72 271-279 FLWGPRALV 73 MAGE-4 230-239 GVYDGREHTV 74GVYDGRIHTV 75 MAGE-C2 336-344 ALKDVEERV 76 Mdm2 81-88 LLGDLFGV 77MELAN-A/ 26-35 EAAGIGILTV 78 MART-1 AAAGIGILTV 79 ALAGIGILTV 80VVAGIGILAI 81 27-35 LAGIGILTV 82 ALGIGILTV 83 MOG 210-218 TLFVIVPVL 84mouse TERT 545-553 QLLRSFFYI 85 797-806 SLFDFFLHFL 86 981-990 YLQVNSQTV87 GnT-V VLPDVFIRC 88 VLPDVFIRCV 89 NPM-1 183-191 AIQDLCLAV 90 AIQDLCVAV91 NY-BR-1 158-167 LLSHGAVIEV 92 960-968 SLSKILDTV 93 1365-1373LLKEKNEEI 94 NY-ESO-1 85-96 SRLLEFYLAMPF 95 86-94 RLLEFYLAM 96 108-116SLAQDAPPL 97 127-135 TVSGNILTI 98 157-165 ALLMWITQC 99 SALMWITQC 100SLAMWITQC 101 SLLAWITQC 102 SLLMAITQC 103 SLLMWATQC 104 SLLMWIAQC 105SLLMWITAC 106 157-166 SLLMWITQCF 107 157-167 SLLMWITQCFL 108 157-170SLLMWITQCFLPV 109 158-166 LLMWITQCF 110 158-167 LLMWITQCFL 111 159-167LMWITQCFL 112 161-169 WITQCFLPV 113 p53 264-272 LLGRNSFEV 114P. aeruginosa 125-133 LAGIGILIV 115 probable sulfate  transporter PRAME300-309 ALYVDSLFFL 116 proteinase 3 169-177 VLQELNVTV 117 RHAMM 165-173ILSLELMKL 118 Rab-38 49-58 KVLHWDPETV 119 50-58 VLHWDPETV 120 SSX2 41-49KASEKIFYV 121 KASEKITYV 122 103-111 RLQGISPKI 123 ALQGISPKI 124ALQGASPKI 125 ALQGISAKI 126 ALQGISPAI 127 SSX4 41-49 KSSEKIVYV 128Surviving  96-104 LTLGEFLKL 129 LMLGEFLKL 130 TERT 540-548 ILAKFLHWL 131Tyrosinase 9-Jan. MLLAVLYCL 132 369-377 YMDGTMSQV 133 Transaldolase168-176 LLFSFAQAV 134 vaccinia virus 60-68 CLTEYILWV 135 C16/B22 WT1126-134 RMFPNAPYL 136 235-243 CMTWNQMNL 137 HLA- Influenza M1 27-35RLEDVFAGK 138 A*0301 influenza NP 265-273 ILRGSVAHK 139 HLA-influenza M1 58-66 GILGFVFTL 140 A*2301 HLA- HER2/neu 63-71 TYLPTNASL141 A*2402 HIV nef 135-142 RYPLTFGW 142 MAGE-4 143-151 NYKRCFPVI 143NY-ESO-1 158-166 LLMWITQCF 144 162-170 ITQCFLPVF 145 SAGE 715-723LYATVIHDI 146 TERT 324-332 VYAETKHFL 147 Tyrosinase 206-214 AFLPWHRLF148 WT1 235-243 CMTWNQMNL 149 417-425 RWPSCQKKF 150 HLA- influenza M158-66 GILGFVFTL 151 A*3101 HLA- gp100/Pmel 17 182-191 HTMEVTVYHR 152A*6801 MAGE-3 115-123 ELVHFLLLK 153 HLA- hCMV pp65 417-426 TPRVTGGGAM154 B*0702 HIV nef 129-138 TPGPGVRYPL 155 NY-ESO-1 60-72 APRGPHGGAASGL156  98-109 TPMEAELARRSL 157  98-110 TPMEAELARRSLA 158 HLA- influenza M158-66 GILGFVFTL 159 B*1302 HLA- NY-ESO-1 88-96 LEFYLAMPF 160 B*1801 HLA-EBV NA-4 488-496 AVLLHEESM 161 B3501 gp100/Pmel 17 630-638 LPHSSSHWL 162hCMV pp65 123-131 IPSINVHHY 163 HIV Gag-Pol 774-782 NPDIVIYQY 164MELAN-A/ 26-35 EAAGIGILTV 165 MART-1 EPAGIGILTV 166 EAAGIGILTY 167EPAGIGILTY 168 NY-ESO-1  92-100 LAMPFATPM 169  92-104 LAMPFATPMEAEL 170 93-104 AMPFATPMEAEL 171  94-102 MPFATPMEA 172  94-104 MPFATPMEAEL 173 96-104 FATPMEAEL 174 116-123 LPVPGVLL 175 Tyrosinase 312-320 LPSSADVEF176 HLA- influenza M1 58-66 GILGFVFTL 177 B3503 NY-ESO-1  92-100LAMPFATPM 178  92-104 LAMPFATPMEAEL 179  93-104 AMPFATPMEAEL 180  94-102MPFATPMEA 181  94-104 MPFATPMEAEL 182 HLA- importin-α2/ 204-212GAVDPLLAL 183 Cw*0304 karyopherin NY-ESO-1  92-100 LAMPFATPM 184  92-104LAMPFATPMEAEL 185  96-104 FATPMEAEL 186 HLA- influenza M1 58-66GILGFVFTL 187 Cw*0702 HLA- CEA 694-702 GVLVGVALI 188 A*0201 GLLVGVALI189 GVLVGVALV 190 GLLVGVALV 191 EBV IE63 259-267 GLCTLVAML 192 193gp100/Pmel 17 209-217 ITDQVPFSV 194 IMDQVPFSV 195 hCMV pp65 495-503NLVPMVATV 196 HER2/neu 369-377 KIFGSLAFL 197 influenza M1 58-66GILGFVFTL 198 MAGE-10 254-262 GLYDGMEHL 199 Mdm2 81-88 LLGDLFGV 200MELAN-A/ 26-35 EAAGIGILTV 201 MART-1 NY-ESO-1 157-165 SLLMWITQC 202SLLMWITQA 203 p53 264-272 LLGRNSFEV 204 proteinase 3 169-177 VLQELNVTV205 RHAMM 165-173 ILSLELMKL 206 SSX2 41-49 KASEKIFYV 207 KASEKITYV 207KASEKITYV 208 WT1 126-134 RMFPNAPYL 209 HLA- DCT/TRP-2 180-188 SVYDFFVWL210 A*0201 α1 HERV 65-74 SILQDLNNFV 211 α2 H-2Kb influenza M1 58-66GILGFVFTL 212 α3 MELAN-A/ 26-35 ELAGIGILTV 213 MART-1 ELAGIGILV 214NY-ESO-1 157-165 SLLMWITQC 215 157-166 SLLMWITQCF 216 157-167SLLMWITQCFL 217 158-166 LLMWITQCF 218 158-167 LLMWITQCFL 219 159-167LMWITQCFL 220 161-169 WITQCFLPV 221 HLA- EBV IE63 259-267 GLCTLVAML 222A*0201 MELAN-A/ 26-35 ELAGIGILTV 223 MART-1 NY-ESO-1 157-165 SLLMWITQA224 225 MHC Protein Position Peptide 226 H-2K^(d) HLA-A2 194-203RYLENGKETL 227 HLA-Cw3 194-203 RYLKNGKETL 228 HER2/nue 63-71 TYLPTNASL229 influenza HA 204-212 LYQNVGTYV 230 mouse ERK2 136-144 QYIHSANVL 231P. berghei CS 252-260 SYIPSAEKI 232 SYIPSAEK(ATTO)I 233 SYIPSAEK(ABA)I234 Dap[Cy5]-YIPSAEK(ABA)I 235 SYILSAEK(ABA)I 236 SEQ ID MHC ProteinPosition Peptide NO SYIASAEK(ABA)I 237 H-2D^(b) GFP 117-125 DTLVNRIEL238 118-125 TLVNRIEL 239 Gp100/Pmel 17 25-33 KVPRNQDWL 240 HPV16 E749-57 RAHYNIVTF 241 HPV16 L1 165-173 AGVDNRECI 242 influenza NP 366-374ASNENMDAM 243 (1968) influenza NP 366-374 ASNENMETM 244 (1976)influenza PA 224-233 SSLENFRAYV 245 influenza PB1 62-70 LSLRNPILV 246LCMV GPC 33-41 KAVYNFATC 247 KAVYNFATA 248 276-286 SGVENPGGYCL 249LCMV NP 396-404 FQPQNGQFI 250 mouse HY 738-746 KCSRNRQYL 251 mouse gp10025-33 EGSRNQDWL 252 mouse Spas-1 H8 244-252 STHVNHLHC 253 NY-ESO-1 86-94RLLEFYLAM 254 H-2D^(k) MPyV MT 389-396 RRLGRTLL 255 H-2D^(d) HIV env309-318 RGPGRAFVTI 256 NY-ESO-1 81-88 RGPERSLL 257 H-2K^(b) chicken258-265 SIINFEKL 258 ovalbumin DCT/TRP-2 180-188 SVYDFFVWL 259E. coli β-gal  97-104 DAPIYTNV 260 H4 87-93 VVYAFKR 261 influenza PB1703-711 SSYRRPVGI 262 LCMV GPC 34-41 AVYNFATC 263 LCMV NP 205-212YTVKYPNL 264 MHV S 598-605 RCQIFANI 265 NY-ESO-1 87-94 LLEFYLAM 266SV40 LT 404-411 VVYDFLKC 267 H-2L^(d) MLV gp70 AH-1 138-147 SPSYVYHQF268 H-2QAI mouse H2-L/  3-11 AMAPRTLLL 269 Qdm TL T3b TL

TABLE 2Exemplary MHC class II molecules and antigenic peptides useful for thegeneration of reversible multimers. SEQ ID MHC Protein Position PeptideNO HLA-DP*0401 adenovirus hexon 911-925 DEPTLLYVLFEVFDV 271 CD74/HLA-DR103-117 PVSKMRMATPLLMQA 272 invariant γ-chain MAGE-3 111-125RKVAELVHFLLLKYR 273 243-258 KKLLTQHFVQENYLEY 274 NY-ESO-1 157-170SLLMWITQCFLPVF 275 157-180 SLLMWITQCFLPVFLAQPP 276 SGQRR tetanus toxin947-960 FNNFTVSFWLRVPK 277 HLA-DQ*0601 influenza HA 57-76QILDGENCTLIDALLGDPQ 278 D gp100/Pmel 17 175-189 GRAMLGTHTMEVTVY 279MELAN-A/ 25-36 EEAAGIGILTVI 280 MART-1 26-35 EAAGIGILTV 281 HLA-DR*0101CD74/HLA-DR 103-117 PVSKMRMATPLLMQA 282 invariant γ-chain influenza HA306-318 PKYVKQNTLKLAT 283 MAGE-3 267-282 ACYEFLWGPRALVETS 284 NY-ESO-187-98 LLEFYLAMPFAT 285 123-137 LKEFTVSGNILTIRL 286 HLA-DR*0401CD74/HLA-DR 103-117 PVSKMRMATPLLMQA 287 invariant γ-chain gp100/Pmel 1744-59 WNRQLYPEWTEAQRLD 288 influenza M1 61-72 GFVFTLTVPSER 289influenza NP 206-229 FWRGENGRKTRIAYERM 290 CNILKGK NY-ESO-1 119-143PGVLLKEFTVSGNILTIRL 291 TAADHR H-2IA^(b) chicken ovalbumin 323-339ISQAVHAAHAEINEAGR 292 mouse DCT/TRP-2 110-124 KFGWSGPDCNRKKPA 293LCMV Pre-GP-C 61-80 GLNGPDIYKGVYQFKSVE 294 FD MoMLV env 120-138EPLTSLTPRCNTAWNRLK 295 L mouse TRP-1 420-434 ADIYTFPLENAPIGH 296

Additional MHC molecules useful according to aspects of this inventioninclude, but are not limited to MHC molecules comprising a mutantHLA-A*0201 chain, e.g. a chain comprising a D227K, a T228A, a D227K, aT228A, a T233A, and/or A245V mutation. Additional MHC molecules usefulaccording to aspects of this invention further include, but are notlimited to HLA-A*1101, HLA-A*3001, HLA-A*3004, HLA-B*0801, HLA-B*2705,HLA-B*5101, HLA-Cw*0303, HLA-Cw*0401, HLA-Cw*0602, HLA-Cw*1402, H-2IAd,and H-2IEd molecules.

In some embodiments, chimeric MHC class I multimers are provided, forexample, multimers in which the comprised heavy chains are in part humanand in part murine. Further, in some embodiments, peptides comprisingmodified amino acid residues are provided, for example, ABA,4-azidobenzoic acid, or Dap, diamino-propionic acid. In someembodiments, peptides are provided that comprise or are conjugated tolow molecular weight fluorescent dyes (see, e.g. www.attotech.com/), forexample, for flow cytometry analysis.

Example 3

Universally applicable methods for the preparation of immunopure MHCII-peptide staining reagents are provided herein. A method for isolationof MHC II molecules that have stably bound a peptide of interestconjugated to a tag, which, in some embodiments, can subsequently beremoved, for example, by cleavage of a linker connecting the tag to thepeptide. Further, a method is provided that allows gentle purificationof fragile “empty” (without nominal peptide cargo) His tagged MHC IImolecules by affinity chromatography on Ni²⁺ nitrilotriacetic acid (NTA)columns. After isolation of correctly peptide loaded MHC II-peptidecomplexes these can be directly converted to multimers by reaction withNTA_(n) conjugated phycobilins (e.g. phycoerythrin) or quantum dots(Qdots).

Abbreviations used in this example include BSP (biotinylation sequencepeptide), DR1 (DRB1*0101); DR4 (DRB1*0401); ESO (NY-ESO-1); GFC (gelfiltration chromatography); HA (influenza hemagglutinin); LZ (leucinezipper); NPβA (3-(2-nitrophenyl)-(3-alanine); NTA (nitrilotriaceticacid); PE (phycoerythrin); and pY (phospho-tyrosine).

While MHC I-peptide complexes can be obtained by peptide drivenrefolding in good yields and high purity, soluble recombinant MHC classII proteins cannot and are typically produced by insect expressionsystems, e.g. Drosophila S2 cells or baculovirus and sf9 cells (1).Deletion of the transmembrane (TM) domains of the α and β chains resultsin their dissociation, which is re-established by addition of leucinezippers. In some embodiments, for multimer (e.g., “tetramer”) formation,a biotinylation sequence peptide (BSP) sequence is added after theleucine zipper (e.g., after the acidic zipper) and enzymaticbiotinylation and tetramerization is performed as for MHC I-peptidemultimers (1-5). In some embodiments, “empty” (without nominal peptidecargo) MHC II molecules are isolated from culture supernatants byimmunoaffinity chromatography and subsequently loaded with a peptide ofinterest. The efficiency of peptide loading depends on its bindingstrength to the MHC II molecule; if it is below a threshold, peptideloading is inefficient and the resulting complexes of limited stability.If this strategy is not feasible, peptides can be tethered to theN-terminus of the β chain via a flexible linker (6). This strategy worksfor some, but not all, complexes. Also, although the peptide is part ofthe molecule, in the case of weak binding peptides there is no knowingwhether or not it is correctly bound in the peptide binding groove.

The staining of antigen-specific CD4+ T cells often is weak and thefrequency of stained cells ex vivo very low, usually necessitating priorin vitro peptide stimulation to permit conclusive detection. There aresignificant differences in multimer staining of CD8+ and CD4+ T cells,such as 1) the staining with MHC II multimers is usually higher at 37°C. than at lower temperatures, which is not the case for MHC I multimers(2, 3, 7); 2) Efficient CD4+ T cell staining requires longer incubationperiods, which is explained, at least in part, by accumulation of MHC IImultimers by endocytosis; therefore agents that affect cell vitality andcytoskeleton function inhibit CD4+ T cell staining (7, 8). 3) Theavidity of MHC II multimer binding is usually lower than the binding ofMHC I multimers, mainly because CD8 greatly strengthens MHC-peptidebinding to CD8+ T cells, whereas CD4 does not (9, 10). MHC II multimerstaining therefore typically requires higher concentrations (up to 100nM, i.e. about 50 μg/ml) (2-5).

While MHC I-peptide complexes obtained by refolding are highly pure andconformationally uniform, MHC II-peptide complexes obtained by peptideloading of “empty” MHC II proteins or containing tethered-on peptidesoften are not, which can seriously impair MHC II multimer staining. Thecommonly used purification of MHC II molecules by immuno-affinitychromatography is not only tedious and expensive, but prone to yieldprotein denaturation. This decreases the fraction of MHC II moleculesthat can be loaded with a given peptide and thus the active reagentfraction of multimers prepared with these monomers (FIG. 26). We havepreviously demonstrated that the quality of MHC II multimers can bedramatically increased when these are produced with molecularly definedmonomers, i.e. monomers that contain only the peptide of interest (2,3). In these studies added a histidine tag (His₆) N-terminally of thepeptide of interest and after peptide loading isolated correctly loadedcomplexes by affinity chromatography on Ni²⁺ NTA (nitrilotriacetic acid)columns. While this method is efficient, it has the disadvantage thatthe peptides are modified with a His tag, which in some cases may affecttheir binding to MHC molecules and/or their T cell recognition.

Here we report that molecularly defined MHC II-peptide complexes can beisolated by anion exchange chromatography when adding an acidic tag onthe peptide of interest.

After purification this tag can be removed by means of a photo-cleavablelinker. This allowed the use of His tagged MHC II proteins which offeredtwo important advantages: 1) the protein can be purified by gentleaffinity purification on Ni²⁺-NTA columns, thereby significantlyreducing denaturation of fragile “empty” MHC II molecules. 2) Afterpeptide loading and isolation of immunopure, bona fide (i.e. withoutpeptide tag) MHC II-peptide monomers can be directly converted inmultimers. In addition we report that enzymatic de-sialylation of cellsincreases their MHC II multimer staining by several-fold, which is asimple means to obtain better staining results.

Peptide Synthesis

Protected amino acids and tentagel resin for solid phase peptidesynthesis were obtained from Rapp-polymere (Tubingen, Germany and BachemAG, Bubendorf, Switzerland). The coupling reagents TBTU and HOBt werepurchased from Multisynthec (Witten, Germany), Cy5.5-maleimide from GEHealthcare and Fmoc-3-amino-(2-nitrophenyl)-propionic acid from PeptechCorporation (USA). Reverse phase HPLC analyses were performed on aWaters system consisting of two Waters 515 pumps and a Waters 996photodiode array detector. The purity of all peptides was assessed byanalytical HPLC. Analytical HPLC columns (Uptisphere 5 μm C₁₈ particles,250×4.6 mm) was eluted at flow of 1 ml/min, and semi preparative HPLCcolumns (Kromasil 15 μm C₁₈ particles, 250×20 mm) at 3 ml/min, with alinear gradient of acetonitril rising in 1 h from 0 to 75% on 0.1% TFAin H₂O. The mass of purified peptides was measured by mass spectrometry(MS) on a MALDI-TOF mass spectrometer.

Peptide syntheses were performed using 2 equiv of N-α-Fmoc-protectedamino acid relative to the resin loading, activated in situ with 2 equivof TBTU, 2 equiv of HOBt and 4 equiv of DIEA in DMF (10 ml/g resin) for1 h. Coupling completion was verified by the Kaiser test. N-α-Fmocprotecting groups were removed by treatment with a piperidine/DMFsolution (1:4) (10 ml/g resin) for 5 min. Crude peptides were obtainedby treating the resin with a solution of TFA/H₂O/TIPS (92.5/2.5/5) for 3h at ambient temperature. Peptides were precipitated with addition ofanhydrous ether, filtered off, dissolved in water, lyophilized andpurified by reverse phase HPLC. For the synthesis of Cy5.5 labelledpeptides, precursors peptide containing a free thiol were dissolved inDMSO at 10 mM and reacted with one equivalent of Cy5.5-maleimide. After2 h of stirring the mixture was directly subjected to bysemi-preparative HPLC.

Photocleavage Under UV Irradiation

Peptides containing the photocleavable amino-acid NPβA in water (50 μM)or MHC II-peptide complexes in PBS (2-20 μM) and irradiated in open 96well plates with a UV lamp (Vilber Luormat) containing two 15 W mercuryfluorescent tubes emitting at 365+/−40 nm at a distance of 10 cm.Samples irradiated for different periods of time were directly analyzedby HPLC and mass spectroscopy.

MHC II-peptide complexes

Extracellular coding parts of DR alpha and beta chains were PCRamplified using ctttagatctcgaccacgtttcttggagc (SEQ ID NO: 297) as the 5′primer and ctttgaattccttgctctgtgcagattcag (SEQ ID NO: 298) as the 3′primer from cDNA preparations (Qiagen) of total RNA extracted from humanPBMCs, digested with appropriate restriction enzymes and cloned in pMT ABiP/V5/His vector-derived cassette (Invitrogen) containing sequences forappropriate leucine zippers and the AviTag (Avidity). The AviTag(GLNDIFEAQKIEWHE, SEQ ID NO: 299) was encoded in an oligo (CTTT CTG GATATC TCA TTC GTG CCA TTC GAT TTT CTG AGC CTC GAA GAT GTC GTT CAG ACC GCCACC, SEQ ID NO: 300) used to extend the basic leucine zipper sequence(TTAPSAQLKKKLQALKKKNAQLKWKLQALKKKLAQ, SEQ ID NO: 301) separated by aflexible linker (GGGSGGS, SEQ ID NO: 302). Oligos for introduction of asingle His6 (CTTTGATATCTCAATGATGGTGATGATGGTGGCCGGTGCGCTGAGCCAGTTCCTTTTCC, SEQ ID NO: 303) or a double His6(CTTTGATATCTCAGTGGTGGTGGTGGTGGTGGCTGCCGCTGCCGCCGCCGCTGCCGCCGCCATGATGGTGATGATGGTGGCCGGTGCG, SEQ ID NO: 304) were designed tofollow the acidic leucine zipper (TTAPSAQLEKELQALEKENAQLEWELQALEKELAQ,SEQ ID NO: 305) on the DRα chain separated by a flexible linkermentioned above. All constructs were verified by sequencing.

Soluble “empty” DR1 (DRA, DRB1*0101), DR4 (DRA, DRB1*0401) and DR52b(DRA, DRB3*0202) molecules were produced in Drosophila melanogaster D.Mel-2 cells (a serum-free medium adapted variant of S2 cells) grown inSf900 II serum-free medium (Invitrogen) at room temperature (22-26° C.).Cells were simultaneously transfected with three plasmids (the plasmidfor DRA, DRB and pBS-PURO, a plasmid conferring puromycin resistance (agift from K. Karjalainen, Nanyang Technological University, School ofBiological Sciences) using Cellfectin (Invitrogen), Singapore). For DR1and DR4, a population of transfected cells was used, whereas for DR52bhigh-yielding clones were obtained by limiting dilution. The cells weregrown in roller bottles (BD Falcon) rotating at 6 rev/min at roomtemperature to 5-10*10⁶/ml and protein production was induced byaddition of 1 mM CuSO₄ for 3-5 days. The yields of purified protein were2-5 mg per liter of medium.

For immuno-affinity purification a column was used containing Sepharose4B coupled with L243 (anti-DRalpha) antibody. Soluble “empty” DRs wereeluted with 50 mM glycine/HCl buffer pH 11.5 and the eluate wasimmediately brought to pH 8.0 by addition of 2 M Tris HCl pH 6.8. Forpeptide loading soluble “empty” molecules were brought to pH 5.5 (DR1and DR4) or pH 6.0 (DR52b) by addition of 100 mM citric acid andincubated with a peptide of interest (1-5 μM final concentration) for 24h at 28° C. (DR52b) or 37° C. (DR1, DR4) in the presence of 0.2% octylβ-D-glucopyranoside (Sigma), protease and phosphatase inhibitorcocktails (Roche). In some cases peptide loaded DR molecules werebiotinylated with recombinant BirA biotin transferase (Avidity)according to the supplier's recommendations. Complexes were purified byGFC on a Superdex S200 column. (GE Healthcare Life Sciences). In thecase of MHC II-peptide complexes containing His tagged peptides (e.g.DR4 peptide-NPβA-SGSGHHHHHH, SEQ ID NO: 306), samples were passesthrough a HisTrap HP column (GE Healthcare Life Sciences), which afterwashing was eluted with 200 mM imidazole, which subsequently was removedby GFC in PBS on a Superdex S200 column (GE Healthcare Life Sciences) orby ultrafiltration. In the case of complexes containing Cy5.5 or pY-D₄tagged peptides samples were dialyzed in 5 mM Tris-HCl pH 9.0, 50 mMNaCl and loaded on a Mono Q 5/50 GL column (GE Healthcare LifeSciences), which was eluted with a NaCl gradient rising win 30 min from0 to 0.5 M NaCl on 20 mM Tris, pH 9.0 at a flow rate of 1 ml andcollecting Purified MHC II-peptide complexes were concentrated on anAmicon Ultra-4 filter concentrator, 10,000 MWCO (Millipore) to 1-2 mg/mlas assessed by the Bradford protein assay (Bio-Rad). Degree ofbiotinylation and purity were assessed by the avidin shift assay and wasroutinely >90%. Briefly, two different amounts of biotinylatedconcentrated complexes (typically 2 and 5 μg) are mixed or not with 10μg of avidin (Pierce) and run on 12% SDS-PAGE (non-boiling,non-reducing). After staining in Gelcode blue (Pierce), gels are scannedand quantified using the ImageQuant TL software (GE Healthcare LifeSciences).

ELISA Assays

For monitoring of Cy5.5 tagged peptides 10 μl aliquots of fractions weresupplemented (10×) with blocking buffer (PBS, 1% BSA, 0.5% Tween 20) andloaded in 1:2 serial dilutions (100 μl/well) in blocking buffer into twowells of 96 well plates (MaxiSorp, Nunc), one of which was coated withanti-DR antibody L243, and the other with anti-Cy5.5 antibody (Sigma).After washing bound complexes were revealed with a biotinylated anti-BSPantibody (a gift from Dr. Gennaro DiLibero, Basel University Hospital)and a secondary antibody conjugated with streptavidin-alkalinephosphatase (Sigma). The plates were developed withp-nitrophenly-phosphate (pNPP) SigmaFAST substrate (Sigma) andabsorbance read at 405 nm on an ELISA plate reader. The concentration ofHis₆ tag was assessed likewise, i.e. biotinylated MHC II-peptidecomplexes were trapped on streptavidin-coated plates (Maxisorp, Nunc)and His tag detected by means of Ni-NTA alkaline phosphatase(His-Detector, KPL).

Peptide Competition Assay

For each test peptide eight wells of a 96-well plate (Maxisorp, Nunc)were filled with 50 ul a citrate saline buffer (50 mM citrate, 200 mMNaCl, pH 5.5) containing 1 μg recombinant empty DR1 protein (1 μg), 0.2%octyl-glucoside, complete protease inhibitors (Roche) and 0.2 μMbiotin-HA₃₀₆₋₃₁₈peptide. Competitor peptides were added to finalconcentrations of 1000, 300, 100, 10, 3, 1, 0.3 and 0.1 μM. Afterovernight incubation at 37° C. the samples were diluted 4-fold with PBSsupplemented with 0.1% BSA and 0.05% Tween 20 and 100 μl applied into 96well plates previously coated with L243 antibody (2 μg/ml); after 1 h ofincubation at room temperature the plates were washed, incubated withstreptavidin-alkaline phosphatase (Sigma) (1:10,000); after 1 h theplates were washed and developed with pNPP SigmaFAST substrate (Sigma)and absorbance read at 405 nm.

Cells Under Study

DR4-restricted, HA₃₀₆₋₃₁₈-specific CD4+ T cell clones were obtained bylimiting dilution cloning of a DR4 HA₃₀₆₋₃₁₈ multimer-sorted populationof a CD4+ T cell line generated from PBMC of a healthy donor (HD137)that was propagated by stimulation with 2 μM HA₃₀₆₋₃₁₈ peptide andγ-irradiated CD4⁻ PBMC in RPMI-1640, 10% human serum AB, supplementedwith 100 U/ml of hIL-2. The clones were propagated byphyto-hemagglutinin (PHA) (Oxoid) stimulations every 2-3 weeks.DR1-restricted, ESO-specific CD4+ T cell lines were derived andmaintained as described previously (3). ESO-specific CD4+ cells fromA2/DR1 mice were obtained as follows: groups of mice (n=4-5) wereimmunized by injections (s.c. at the base of the tail) of 50 μl ofemulsion containing the indicated peptides (50 μg of peptide emulsifiedin 50 μl PBS and 50 μl complete Freund's adjuvant (DIFCO); 7-8 dayslater the draining lymph nodes were removed and homogenized by passingthrough a cell strainer (BD Falcon) to obtain single cell suspensionsfor further analysis.

Tetramers and Staining

Biotinylated DR-peptide complexes were multimerized by mixing with smallaliquots of streptavidin-PE (Invitrogen) up to the calculated 4:1stoichiometric equivalent. Cells were stained in 50 μl of FACS buffer(PBS, 0.5% BSA, 2 mM EDTA, 0.05% sodium azide) for 1 hour at 37° C. Inthe case of neuraminidase treatment, cells were incubated with 0.03 U/mlof neuraminidase from V. cholerae (Roche) in complete medium and washedtwice prior to tetramer staining. In some experiments fluorescentantibodies (e.g. anti-CD4) were added after multimer staining andincubated at 0-4° C. for 15 min. After 2 washes the cells were suspendedin 300 μl buffer and analyzed by flow cytometry on a FACSCalibur (BD).Propidium iodide (Invitrogen) was added just before acquisition forexclusion of dead cells. Human cells were fixed in 2% formaldehyde(Polysciences) in FACS buffer analysis. Data analysis was performed withFlowJo 7.6 software (TreeStar).

Preparation and Evaluation of Immunopure Biotin-Streptavidin Multimers

In order to produce immunopure MHC II-peptide complexes, we used a His₆tag linked via a short flexible GSG linker (H₆-GSG-) to the N-terminusof the peptide of interest, which allowed isolation of pure complexes byaffinity chromatography on Ni²⁺ NTA columns (2, 3). Although in thesestudies the added His₆ tag had only modest effects on T cell recognitionand/or MHC binding, this may not be the case in other applications. Inorder to avoid this, we introduced 3-(2-nitrophenyl)-β-alanine (NPβA)between the H₆-GSG moiety and the peptide of interest (FIG. 20). Thistag (H₆-GSG-NPβA) can be added N- or C-terminally to the peptide ofinterest (FIG. 21). Upon UV irradiation at 365 nm the NPβA residue iscleft, resulting in the removal of the His tag after its use foraffinity purification of MHC II-peptide complexes (FIG. 21A). NPβA hasbeen used for the preparation of conditional MHC I-peptide ligands andits usefulness is well documented (11, 12). As shown for immunopureDR4/HA₃₀₆₋₃₁₈ complexes carrying the His tag either N-terminally(His₆-GSG-NPβA-HA₃₀₆₋₃₁₈ or C-terminally (HA₃₀₆₋₃₁₈ NPβA-GSG-H₆) the tagis cleft rapidly upon UV irradiation at 365+/−40 nm, with 50% cleavageachieved after 0.51 and 0.57 min, respectively (FIGS. 21B, C). It shouldbe noted that this cleavage is only about 90% complete; higher cleavageyields can be achieved by using two NPβA groups (12).

When the His tag is added C-terminally at the peptide, it emerges afterphoto-cleavage as an amide, i.e. its C-terminal carboxyl group is anamide (FIG. 21A). Conversely when the tag is added N-terminally thepeptide carries after photolysis an N-terminal2-nitroso-phenacetyl-β-acetoyl moiety (FIG. 21A). To compare thestaining of immunopure DR4-HA tetramer prepared by using either the N-or C-terminal His tag with and without photo-cleavage, we performed 37°C. binding isotherms on four DR4-restricted, HA-specific Th1 clones. Onall four clones the binding of immunopure multimers was considerablymore avid as compared to the conventional DR4/HA multimer (FIGS. 22A-D).The differences were particularly striking on the low affinity clones 15and 8. The multimer containing the N-terminally tagged HA peptide(His₆-GSG-NPβA-HA₃₀₆₋₃₁₈) exhibited the most efficient binding on allclones tested. After photolytic removal of the His tag the stainingefficiency decreased on clone 2, less on clones 9 and 15, but remainedunchanged on clone 8. Conversely, multimers containing the C-terminallytagged HA peptide (HA₃₀₆₋₃₁₈-NPβA-GSG-H₆) exhibited modestly increased(clone 2) or the same binding (clones 8, 9, 15) compared to themultimers in which the tag was removed by photolysis. Importantly, afterUV irradiation mediated removal of the tag both multimers (i.e. thosecarrying His₆-GSG-NPβA-HA₃₀₆₋₃₁₈ and HA₃₀₆₋₃₁₈ NPβA-GSG-H₆) stained allfour clones very similar, arguing that the small modifications of thepeptide by the photo-cleavage little affect multimer staining (FIGS.21A, 22A-D).

Taken collectively these results demonstrate that purification ofcorrectly loaded MHC II-peptide monomers very significantly increasesmultimer binding. As we have shown previously this is critical for thedetection of antigen-specific CD4+ T cells directly ex vivo (2, 3).Moreover, our results indicate that the presence of an N-terminal His₆tag can artificially increase multimer staining (FIG. 22). This isexplained, at least in part, by increased peptide binding to therestricting MHC II molecule (FIG. 27 and refs. 2, 3). This we observedfor the N-terminally, but not (or barely) for the C-terminally Histagged peptide (FIG. 22). This observation is consistent with reportsshowing that adding tags N-terminal to MJC II binding peptides such asinvariant chain derived KEY tags (13, 14) or photoreactive groups (15)can considerably increase their binding. In newly synthesized MHC IImolecules the peptide binding site is occupied by the CLIP sequence ofthe invariant chain (II), which on its way to the plasma membrane“clings” to invariant regions, mainly of the β2 domain, situated infront of the N-terminal portion of the peptide binding groove (16). Onemight argue that thus increased peptide binding could be a means toincrease multimer staining. This, however, is risky as this engagementof this invariant region on MHC II proteins may provoke extendedconformational changes, which may alter the fidelity of the multimerstaining (17). We therefore strongly advocate the use of photo-cleavabletags, which can be removed after purification of correctly loaded MHCII-peptide monomers.

Preparation of Immunopure DR4-HA₃₀₆₋₃₁₈ Monomers Using Cy5.5 TaggedPeptides

A major shortcoming of conventional MHC II multimer synthesis is thatone does not know what fraction of the MHC II molecules have bound thepeptide of interest. This fraction can be highly variable, depending onthe MHC II molecule, its purification and on the peptide and loadingprocedures. Because the effective multimer fraction decreases with thefraction of correctly peptide loaded monomers (FIG. 26) a frequent,probably the most frequent failure of poor or undetectable MHC IImultimer staining is inadequate peptide loading. In order to able todirectly monitor MHC II peptide loading, we used Cy5.5 as tag forlabelling antigenic peptides. Cy5.5 (and Cy5) is a blue fluorescent lowmolecular dye that can be coupled to peptides via amides or thioethers(FIG. 23A, www.gelifesciences.com). To establish a proof a principle, weloaded DR4 with Cy5.5 labelled HA₃₀₆₋₃₁₈ peptide (HA₃₀₆₋₃₁₈-GSGC-Cy5.5,SEQ ID NO: 307) and analyzed the reaction mixture by GCF on SuperdexS200 column recording the OD of the eluent at 675 nm (Cy5.5) and 280 nm(protein) (FIG. 23B). From the integrated peak surfaces and the molarextinction coefficients, the ratio of Cy5.5 (i.e. peptide) and protein(i.e. DR4) was calculated to be 0.42, i.e. 42% of the input DR4molecules had bound the blue peptide. Analogous experiments wereperformed with DR52b and Cy5.5 labelled NY-ESO-1 peptide₁₂₃₋₁₃₇, inwhich case peptide loading was remarkably poor (≦10%) and no significantstaining was observed with the corresponding multimers (data not shownand ref. 2).

As for His₆ tagged peptides, the content of Cy5.5 (or Cy5) labelledpeptide in MC II-peptide complexes can also be detected by ELISA, usingCy dye specific antibodies (FIG. 23C). Moreover and importantly Cy5.5contains four negatively charged sulfonyl groups (SO3⁻), i.e. isstrongly acidic. This allows quantitative separation of theCy5.5-peptide loaded DR4 molecules from other DR4 molecules by anionexchange chromatography (FIG. 23D). Although this method allows directassessment of peptide loading efficiency and isolation of immunopure MHCII-peptide complexes, it has disadvantages, such as i) the synthesis ofCy5.5 labelled peptides is expensive and tedious and ii) the removal ofCy5.5 by photolysis by means of a NPβA linker is not feasible due toquenching.

Preparation of Immunopure MHC II-Peptide NTA Multimers Using thepY-D4-Tag

Isolation of “empty” MHC II molecules from culture supernatants byimmuno-affinity chromatography is not only costly and often inefficient,but is a major cause for extensive protein denaturation. In order toobtain efficient MHC protein recovery, high affinity antibodies areused, which for elution require the use of extreme pH (e.g. 11.5) atwhich empty MHC II molecules start to denature. To circumvent this weadded at the DRA chain C-terminal of the leucine zipper a His tag, whichallows gently and universal purification of empty MHC II molecules byaffinity chromatography on commercially available Ni²⁺ NTA columns (FIG.24). This necessitated the use of another tag to be used for thepurification of correctly peptide loaded MHC II-peptide complexes.Encouraged by the finding that a negatively charged tag allowspurification of correctly loaded MHC complexes, we searched for arelated strategy, namely for a negatively charged tag that can bereadily synthesized and then be removed by photolysis. As new tag weused phospho-tyrosine-Asp₄ (pY-D₄), which can be detected by anti-pYantibodies in ELISA and having six negative charges is expected to allowseparation of loaded MHC II molecules by anion exchange chromatography(FIG. 24). Moreover and importantly, after peptide loading, purificationof correctly peptide loaded complexes and removal of the pY-D4 tag byphotolysis, the His tag can be used again to directly form fluorescentstaining reagents by reaction with NTA_(n)-PE or NTA_(n)-Qdots (asdescribed for MHC I-peptide complexes in the first technical report).

To validate the feasibility of this strategy, we passed a supernatantfrom S2 cells secreting soluble His₆ tagged DR4 over a Ni²⁺-NTA column.After washing the column, it was eluted with 200 mM imidazole, wherebyDR4 eluted in a sharp peak, which according to GFC on a Superdex S200column and SDS-PAGE was pure (FIGS. 25A-C). This material was thenloaded with pY-D4-GSG-NPβA-HA₃₀₆₋₃₁₈ peptide and subjected to anionexchange chromatography on Mono-Q column, which was eluted with theindicated NaCl gradient and on the OD of the eluate was monitored at 280nm and the content of DR4 and pY, respectively, of the fractionsdetermined by ELISA (FIG. 25D). This analysis showed that DR4 containingthe acidic pY-D4-GSG-NPβA-HA₃₀₆₋₃₁₈ peptide eluted after DR4 moleculescarrying no or different peptides, similarly as observed for the Cy5.5tagged HA peptide (FIG. 23D). Because phospho-tyrosine a priori issusceptible to enzymatic de-phosphorylation (although with completephosphates inhibitors, e.g. from Roche, this can be prevented) we alsotested para-sulfate-tyrosine (e.g. Y(SO₄)-D₄) as tag, but observedlesser shifts of the acidic peptide containing DR4 complexes (data notshown). In order to further to extend and optimize the separation ofcorrectly loaded MHC II-peptide complexes we have evaluated variationsin the conditions of the anion exchange chromatography (e.g. differentbuffers and NaCl gradients) as well other acidic tags (pY-E₄₋₈) as wellas other MHC II molecules (e.g. DR1 and DR52b) and other peptides (e.g.NY-ESO-1₁₂₃₋₁₃₇). From these experiments it is apparent that correctlyloaded MHC II peptide complexes can be separated from other MHC IImolecules by anion exchange chromatography; however for general usagethe best suitable acidic tags and chromatography conditions may differsomewhat from those described here.

Over a dozen of different reagents were investigated for their abilityto increase MHC II multimer staining. It was determined that briefpre-treatment of the cells with neuraminidase increases multimer bindingby 2-5-fold. This was observed on a range of DR4-restricted, HA-specificCD4+ T cell clones (FIG. 28) and on populations of HA peptide stimulatedPBMC from healthy donors. Because MHC II multimer staining tends to beweak, this observation suggests pre-treatment of the cells withneuraminidase, an enzyme that removes sialic acid residues on cellularsurface proteins (18) increases MHC class II multimer stainingefficiency.

REFERENCES

-   1. Guillaume P, Dojcinovic D, Luescher IF. Soluble MHC-peptide    complexes: tools for the monitoring of T cell responses in clinical    trials and basic research. Cancer Immun. 2009; 9:7.-   2. Ayyoub M, Dojcinovic D, Pignon P, Raimbaud I, Schmidt J, Luescher    I, Valmori D. Monitoring of NY-ESO-1 specific CD4+ T cells using    molecularly defined MHC class II/His-tag-peptide tetramers. Proc    Natl Acad Sci USA. 2010; 107:7437-42.-   3. Maha Ayyoub, Pascale Pignon, Danijel Dojcinovic, Isabelle    Raimbaud, Lloyd J. Old, Immanuel Luescher and Danila Valmori 2010.    Assessment of vaccine-induced CD4 T cell responses to the 119-143    immunodominant region of the tumor-specific antigen NY-ESO-1 using    DRB1*0101 tetramers. Clin. Cancer Res. accepted.-   4. Danke N A, Koelle D M, Yee C, Beheray S, Kwok W W. Autoreactive T    cells in healthy individuals. J. Immunol. 2004; 172:5967-72.-   5. Cecconi V, Moro M, Del Mare S, Dellabona P, Casorati G. Use of    MHC class II tetramers to investigate CD4+ T cell responses:    problems and solutions. Cytometry A. 2008; 73:1010-8.-   6. Cunliffe S L, Wyer J R, Sutton J K, Lucas M, Harcourt G,    Klenerman P, McMichael A J, Kelleher A D. Optimization of peptide    linker length in production of MHC class II/peptide tetrameric    complexes increases yield and stability, and allows identification    of antigen-specific CD4+T cells in peripheral blood mononuclear    cells. Eur J. Immunol. 2002; 32:3366-75.-   7. Cameron T O, Cochran J R, Yassine-Diab B, Sékaly R P, Stern L J.    Cutting edge: detection of antigen-specific CD4+ T cells by HLA-DR1    oligomers is dependent on the T cell activation state. J Immunol.    2001: 166:741-5.-   8. Wooldridge L, Lissina A, Cole D K, van den Berg H A, Price D A,    Sewell A K. Tricks with tetramers: how to get the most from    multimeric peptide-MHC. Immunology 2009; 126:147-64.-   9. Luescher I F, Vivier E, Layer A, Mahiou J, Godeau F, Malissen B,    Romero P. CD8 modulation of T-cell antigen receptor-ligand    interactions on living cytotoxic T lymphocytes. Nature 1995;    373:353-6.-   10. Hampl J, Chien Y H, Davis M M. CD4 augments the response of a T    cell to agonist but not to antagonist ligands. Immunity 1997;    7:379-85.-   11. Toebes M, Coccoris M, Bins A, Rodenko B, Gomez R, Nieuwkoop N J,    van de Kasteele W, Rimmelzwaan G F, Haanen J B, Ovaa H, Schumacher    T N. Design and use of conditional MHC class I ligands. Nat. Med.    2006; 12:246-51.-   12. Celie P H, Toebes M, Rodenko B, Ovaa H, Perrakis A, Schumacher    T N. UV-induced ligand exchange in MHC class I protein crystals. J    Am Chem. Soc. 2009; 131:12298-304.-   13. Kallinteris N L, Lu X, Blackwell C E, von Hofe E, Humphreys R E,    Xu M. II-Key/MHC class II epitope hybrids: a strategy that enhances    MHC class II epitope loading to create more potent peptide vaccines.    Expert Opin Biol Ther. 2006 6:1311-21.-   14. Kallinteris N L, Lu X, Wu S, Hu H, Li Y, Gulfo J V, Humphreys R    E, Xu M. II-Key/MHC class II epitope hybrid peptide vaccines for    HIV. Vaccine. 2003 21:4128-32.-   15. Luescher I F, Allen P M, Unanue E R. Binding of photoreactive    lysozyme peptides to murine histocompatibility class II molecules.    Proc Natl Acad Sci USA. 1988 85:871-4.-   16. Jasanoff A, Wagner G, Wiley D C. Structure of a trimeric domain    of the MHC class II-associated chaperonin and targeting protein II.    EMBO J. 1998; 17:6812-8.-   17. Rotzschke O, Lau J M, Hofstatter M, Falk K, Strominger J L. A    pH-sensitive histidine residue as control element for ligand release    from HLA-DR molecules. Proc Natl Acad Sci U S A. 2002; 99:16946-50.-   18. Chen X, Varki A. Advances in the biology and chemistry of sialic    acids. ACS Chem. Biol. 2010; 5:163-76.

The entire contents of all of the references (including literaturereferences, issued patents, published patent applications, andco-pending patent applications) cited throughout this application arehereby expressly incorporated by reference for the purposes or subjectmatter referenced herein. In case of a conflict between a referenceincorporated herein and the instant disclosure, the teaching of theinstant disclosure shall control.

Example 4 PE-NTA-A2/Peptide Multimers—a New Type of Staining ReagentBased on Oxime Ligation

To generate PE-NTA multimers, 1 nmol Phycoerythrine (PE) was firstreacted in phosphate buffer, pH 7.4, with 20 mM of sulfo-SFB(sulfo-succinimidyl-formylbenzoate) for 4 h at room temperature. Theresulting activated-PE was subsequently dialyzed in 2 L PBS over 2 daysand then coupled in phosphate buffer (pH 7.2) with a large excess ofH₂N—O-NTA₂ or H₂N—O-NTA₄ aminooxy-containing peptide. After overnightreaction at 4° C., bioconjugates were loaded with Ni²⁺ and dialyzed in 2L PBS over 2 days. PE-NO-NTA₂ or PE-NO-NTA₄ obtained were conjugatedwith A2/peptide-2×His6 complexes to generate biotin SA free PE-NTA₂ orPE-NTA₄ multimers.

Staining is a Function of PE Substitution Degree.

The staining characteristics of the multimers generated via oximechemistry were investigated. FIG. 29A illustrates a scheme of PEactivation with sulfo-SFB and conjugation with H₂N—O-NTA₂ via oximeligation. FIG. 29B: PE (1 nM) was activated with the indicatedconcentrations of sulfo-SFB and conjugated with an excess of NTApeptide. 0.5 μg of PE-NO-NTA₂ were mixed with 5 μg of A2/Flu58-66-2×His6monomers. Flu matrix 58-66-specific BC74cells were stained with 8 nM ofthe different conjugates at room temperature for 30 min and analyzed byflow cytometry. SFB: succinimidyl-p-formylbenzoate.

FIG. 30 shows staining isotherms on Flu stimulated PBMC. FIG. 30A: PEactivated with 1, 10 or 20 mM sulfo-SFB was coupled to NTA₂ peptide viaoxime ligation and subsequently mixed with A2/Flu-2×His6 A2/Flu58-66monomers. Flu matrix peptide stimulated PBMC were stained with differentconcentrations of the conjugates and analyzed by flow cytometry. Thenumbers indicate MFI of specific (above the bar in each graph) andnon-specific (below the bar) staining. The right hand numbers indicatethe input amounts of monomers (upper number) and PE-NTA2 (lower number).FIG. 30B: Data plotted using GraphPad Prism software.

FIG. 31 shows exemplary NTA linkers that were synthesized and tested.The bioNTA4 and oxNTA2 linkers were determined to be of particularinterest for the generation of MHC I and MHC II multimers and were usedfor further testing as described below. The bioNTA₂ and bioNTA₄compounds contain biotin-NTA2 or 4, all others contain N terminalCys(SH)-PEG₂ on NTA₂ or NTA₄ to be used for conjugation withmaleimido-PE. The non-biotin compounds contain N-terminal imines andNTA₂ linked via a PEG₂ Spacer, a PEG₄ spacer, or a dipeptide linker(bottom).

PE conjugates with these linkers were obtained and the performance ofthese conjugates was tested. The conjugates comprised either A2/Flu58-66monomers, which were tested in staining of Flu peptide stimulated PBMC,or comprised DR4/Flu HA306-318 monomers, which were tested in stainingof Flu-Specific CD4+ T cells (FIG. 32). From these initial tests, thebiotin-NTA4 and SA-PE multimers appear to perform similar toconventional BSP multimers, but result in better stainings of both CD8⁺and CD4⁺ T cells. In addition, the multimers described herein are fullyreversible, as explained in more detail elsewhere herein, which allowssorting of antigen-specific T cells without causing activation dependentT cell death. The data presented here indicates that thePEG2-NTA2-comprising PE-NTAmers coupled to PE via oxime bond formationexhibit an improved staining performance as compared to multimersobtained by conjugation via maleimides. This may in par be due to anincreased degree of conjugation associated with the use ofoxime-chemistry.

Comparison of Conventional, PE-Cys-PEG2-NTA2 and PE-HNO-NTA2 MultimersStaining.

FIG. 33A shows the results of an experiment in which cloned, Flu matrix58-66-specific CD8⁺ 81P1 cells were incubated at 20° C. for 30 min withgraded concentration of conventional multimers (circles),PE-Cys-PEG₂-NTA₂ NTAmers (squares), or PE-HNO-NTA₂ NTAmers (triangles).Cell-associated PE fluorescence was then assessed by flow cytometry. Thebinding data of FIG. 33A were subjected to Scatchard analysis (FIG. 33B), and K_(D) and B_(max) were calculated from the results of theScatchard Analysis (FIG. 33C).

Reducing of Background Staining by Milk Supplements.

It was found that the background staining observed when using multimersgenerated via oxime chemistry can be reduced by using a proteinsupplement, for example, milk protein, during the staining procedure(FIG. 34). Cloned, A2/Flu matrix58-66 specific 81P1 cells were incubatedwith a A2/Melan-A—specific clone at 20° C. for 30 min with the indicatedconcentrations of conventional (column 1) or PE-HNO-NTA₂ containingmultimers (columns 2, 3, 4, 5 and 6) in the absence (row 1) or presenceof the indicated concentrations of dried milk powder (rows 2-4). Milkpowder efficiently decreased the background staining of oxime-chemistrymultimers.

Binding Titration of NTAmers to Determine Best Dilution for Ex VivoStaining.

Cloned, Melan A LAU 959 46 (FIG. 35A) or EBV LAU 1013 specific clonedcells (FIG. 35B) were mixed with fresh PBMC and incubated at 20° C. for30 min with graded dilution of PE-NTA2 NTAmer (row 2), SA-PE multimerscontaining SA-PE biotin-NTA₂ (row 3) or SA-PE biotin-NTA₄ (row 4).

Ex vivo staining with BSP and NTA multimers on fresh PBMC was alsocompared. FIG. 36 shows ex vivo flow cytometric analysis of PBMC stainedwith A2/peptide multimers containing BSP (conventional), PE-oxime NTA₂,or SA-PE biotin-NTA₄. PBMCs were obtained from EBV+(BCL 7) individuals(FIG. 36A), CMV+(BCL 8) individuals (FIG. 36B), and from two melanomapatients: LAU 616 (FIG. 36C) and LAU 1164 (FIG. 36D).

Example 5 Use of Desthiobiotin for Purification of Correctly PeptideLoaded MHC II-Peptide Complexes

In some embodiments, multimeric MHC II staining reagents describedherein are generated by loading monomeric MHC molecules with a peptideof interest and purifying correctly peptide-loaded monomers for furtherprocessing and assembly to multimers. For example, in some embodiments,a hexahistidine (His6) tag is added N-terminally at the peptide and thecomplexes are purified on Ni²⁺ NTA columns. In some embodiments, anN-terminal polyacidic tag is used, which allows purification ofcomplexes by anion exchange chromatography. In some embodiments, adesthiobiotin (DTB) tag is N-terminally added to the peptide usingconventional solid phase peptide synthesis (SPPS) and the targetcomplexes are purified on streptactin columns using elution with freeDTB. The column can be completely regenerated by washing with2-(4′-hydroxyazobenzene) benzoic acid (HABA) and the Tris, pH 9.0. Therecovery yields are close to 100%, and pMHC II complexes fully active.

The use of DTB is less complex than using a poly-acidic tag and anionexchange chromatography, and has the advantage that a His tag can beused on the MHC II protein. In some embodiments, this allows i) gentleand universal purification of MHC II proteins from supernatants and ii)diverse conjugate formation based on the His tag-NTA chelate complexconjugation strategies described herein. An overview of an exemplarystrategy for NTA-His tag multimer preparation using DTB is outlined inFIG. 37.

Protein Expression in Drosophila Cells

Serum-free adapted Drosophila melanogaster cell line (D.mel-2) weretransfected concomitantly with two pMT-derived plasmids containingcloned extracellular parts of the a and beta chain of the MHC class IIallele, followed by leucine zipper sequences and terminating with aAvi-Tag (beta chain) or a tandem His-tag (a chain). A pBS-derivedplasmid containing the puromycin resistance gene is co-transfected toallow for antibiotic selection. After selection in 10 μg/ml puromycin(Sigma), cells were cloned by limiting dilution and clones screened forhigh expression. Expression levels in supernatants of DR1, DR4 and DP4proteins with C-terminal tandem His-tags range from 1 to 5 mg/ml asdetermined by ELISA.

Recombinant MHC Class II Protein Purification on IDA Columns

Clarified and 0.22 μm filtered supernatants from 3-5 day conditionedSf900 II SFM media (Invitrogen) from insect cell culture were flowedthrough a column of 25-50 ml Chelating Sepharose FF (GE Healthcare LifeSciences) at 1.5-2 ml/min (previously equilibrated in PBS) and washedwith 10 mM imidazole in PBS. Tandem His-tagged recombinant MHC class IIproteins were eluted with 200 mM imidazole in PBS and the columnregenerated with 20 mM EDTA in 50 mM Tris pH 8.0. The eluate was furtherconcentrated in an Amicon cell concentrator (Millipore) and bufferexchanged with a HiLoad 26/10 gel filtration column (GE Healthcare LifeSciences) against the loading buffer (100 mM sodium citrate pH 6.0).

Peptide Loading and Purification of “Immunopure” Complexes

Loading of purified recombinant MHC class II protein with 100 μMdesthiobiotin (DTB) or polyglutamate-tagged (pY-E8) HA308-318 orNY-ESO-1119-143 peptide at 100 μM was performed at 37° C. for 24 h. Toremove the excess peptide, the sample was passed through the HiLoad26/10 column twice in PBS (for DTB-tagged complexes) or 50 mM Tris pH9.0 (for pY-E8-tagged complexes) and then applied to a StrepTrap HP 1 ml(for DTB-tagged complexes) or a MonoQ 4.6/100 (for pY-E8-taggedcomplexes). After a 5 CV wash, DTB-containing complexes were eluted with50 mM desthiobiotin in PBS (pH corrected to 7.4) while the pY-E8complexes were eluted with a NaCl gradient (0-1 M NaCl in 20 CV) at 400mM NaCl. Flow rates were 1 ml/min for the StrepTrap column and 2 ml/minfor the MonoQ column (both columns from GE Healthcare Life Sciences).The StrepTrap column is regenerated with 1 mM HABA (Sigma) in PBS andequilibrated in 30 CV of PBS. The eluted proteins were diluted with anequal volume of PBS (DTB-tagged peptide) or 50 mM Tris pH 9.0(pY-E8-tagged peptide) before being concentrated to 0.5-1 mg/ml in anAmicon-5 filter concentrator (Millipore). Aliquots of 10-25 μg were snapfrozen in liquid nitrogen and stored at −80° C. Optional tag removal byUV. In case the tag is linked to the peptide with a photocleavableamino-acid derivative (βNPA), it can be removed by irradiation with a 30W UV lamp (365 nm) by irradiating for 10 min in V-bottom 96-well plates(1 μg MHC-peptide complex in 100 ul PBS).

Multimerization

Purified MHC class II-peptide complexes with C-terminal tandem His-tagsare added to NTA-derivatized PE and SA-PE conjugates in a well of aV-bottom 96-well plate, vigorously mixed and incubated at least 2 hoursat 4° C. before use in staining a cell sample.

Use of DTB to Isolate MHC-II Peptide Complexes

The structure of biotin and desthiobiotin are provided in FIG. 38. Wefound out that DTB binds to StreptActin with sufficient affinity topermit efficient retention and can be readily displaced by free DTB.Importantly, StreptActin columns could be completely regenerated bywashing with HABA, which displaces DTB, and subsequently with Tris, pH9.0, which removes HABA. Contrary to previous reports, the regenerationof streptavidin columns after elution with free DTB was incomplete (withHABA and/high or low pH). While streptactin columns are used forpurifying Strep tag-containing molecules and monomer streptamer columnsfor purifying biotinylated molecules, we report here that StreptActincolumns allow efficient purification of DTB tagged molecules andcomplete regeneration of the columns.

Evaluation of DTB Tag Peptide Purification Efficiency

A DTB tagged model peptide was generated and purification performancewas evaluated (FIG. 39). The structure of a low molecular weight coloredpeptide used for quantification of the purification efficiency isdescribed in FIG. 39A. FIG. 39B shows a compilations of the efficienciesof retention, elution and regeneration for 3 different columns. FIG. 39Cshows purification yields on StreptActin superflow high capacity columnfor three consecutive rounds of purification/regeneration. Regenerationbuffer: HABA 0.1 M followed by Tris 0.2 M, pH 9.

DTB tag MHC I monomer purification efficiency was evaluated next (FIG.40). Binding/Washing buffer: Tris 0.1 M, 150 mM NaCl, 1 mM EDTA, pH 8.Elution Buffer: Tris 0.1 M, 150 mM NaCl, 1 mM EDTA, pH 8, 50 mM DTB.Regeneration Buffer: Tris 0.1 M, 150 mM NaCl, 1 mM EDTA, pH 8, 1 mM2-(4′-hydroxyazobenzene) benzoic acid (HABA). For purificationevaluation, 499 μg of pMHC I monomers carrying a DTB C terminal on theheavy chain were applied onto 2 ml of StreptActin High Capacitysepharose (IBA). Flowthrough, washing and elution were quantified by atOD 350 nm measurements (FIG. 41). Elution was with 50 mM DTB with arecovery of 307 μg (85% yield).

Purification of pMHC I DTB complexes on StreptActin sepharose was alsoevaluated. FIG. 42A shows a chromatogram of a DTB-tagged proteinpurified on a StreptActin sepharose column. FIG. 42B shows thepurification parameters for DTB tagged MHC class I-peptide complexes.

An exemplary scheme of DTB-HA peptide loading and purification isdescribed in FIG. 43. An exemplary structure of DTB-HA and thecorresponding DR42×His DTB-HA complexes are depicted in FIG. 43A. Aschematic of the purification and regeneration cycle of DR42×His DTB-HAon a StreptActin sepharose column is illustrated in FIG. 43B. DR4-DTB-HApeptide complexes were generated and purified on a streptactin column.After peptide-loading, DR4-2×His DTB-HA306-318 complexes were passedover a StreptActin column, washed and eluted with 50 nM DTB as indicatedby arrows (FIG. 44A), and the OD of the effluent at 280 nm wasmonitored. Pure DR4-HA complexes eluted in a sharp peak (indicated by abox in FIG. 44A). DR42×His DTB-HA306-318 complexes were analyzed bySDS-PAGE (12%) before (lane 1) and after (lane 2) purification onStreptActin sepharose (FIG. 44B). SDS-PAGE was performed undernon-reducing, non-boiling (NR, NB, left gel image) or reducing andboiling conditions (R, B, right gel image). Each lane was loaded with 10μg protein and the gel was stained with Coomassie blue. DTB-tag basedpurification resulted in highly pure peptide-loaded MHC preparations.Surprisingly, the purity of DTB-tagged peptide-loaded MHC moleculesobtained by StreptActin column purification was observed to be superiorto that of purification of polyanionic peptide tagged MHC molecules,which yield highly pure peptide-loaded MHC molecules.

Staining of CD4+ DR4-Restricted Flu-Specific T Cells with DTB ImmunopureMultimers.

Staining performance of PE-NTA₂, biotin-NTA₄-SA-PE, and conventional BSPmultimers was compared. As described in more detail elsewhere herein,PE-NTA₂ and biotin-NTA₄-SA-PE multimers contain the NTA moieties shownin FIG. 45.

MHC-Peptide NTAmer Preparation

5 μg DR4 2×His DTB-HA complexes were incubated at 4° C. for 2-16 h with1.4 μg PE NTA2, or 2.8 μg SA-PE NTA4 in wells of a V-bottom 96 wellplate; then each incubation was diluted with EDTA-free FACS buffer (0.5%BSA in PBS with 0.05% sodium azide). Staining of cells was performed in50 μl volume at room temperature (RT) or 37° C. for 1-2 h or asindicated. The PE-NTA₂ generated contained an estimated 7-10 monomersper conjugate. The BSP and biotin-NTA₄ multimers were prepared with thesame SA-PE (from Caltag).

Cloned Flu HA₃₀₆₋₃₁₈-specific CD4⁺ T cells (clone 23-1) were incubatedat room temperature (RT) or 37° C. DR4/HA₃₀₆₋₃₁₈ BSP, biotin-NTA₄-SA-PA,or PE-NTA₂ multimers made with DTB-streptactin purified monomers andafter washing were analyzed by flow cytometry (FIG. 46A, “multimer”refers to conventional BSP multimers). The background staining (blackhistograms) was assessed by parallel staining on a DR4-restricted/Flumatrix 61-72-specific clone. Five percent of cloned 23-1 clone cellswere added to fresh PBMC and analyzed in the same manner (FIG. 46B).

HA-specific CD4⁺ T cell clones 9(A) or 8(B) were incubated for 1 hour at37° C. with graded concentrations of DR4/HA₃₀₆₋₃₁₈ multimer (as inprevious experiments), washed and analyzed by flow cytometry (FIG. 47).A DR4-restricted Flu matrix specific clone was used to evaluatenonspecific binding, which was subtracted. Scatchard analysis wasperformed and the K_(D) (dissociation constant) and Bmax (maximalbinding) values are described in the tables on the right.

The PE-NTA₂ multimers exhibited efficient binding/staining, particularlyon clones that exhibited poor BSP multimer staining. Similarly, thebiotin-NTA₄-SA-PE multimers stained with higher efficiency than BSPmultimers. It is important to note, however, that significant clonalvariations were observed.

DR4/HA₃₀₆₋₃₁₈ SA-PE NTA₄ multimers exhibited less background stainingthan PE NTA₂ multimers (FIG. 48). HA peptide-stimulated PBMC wereincubated at room Temperature (A, B) or 4° C., (C). Optimal multimerconcentrations (14 μg/ml for PE NTA₂, 28 μg/ml for SA-PE NTA₄) were usedin (A) and (B). In (C), three different concentrations of the PE NTA₂reagent were compared. Non-stimulated PBMCs were used as a control.

The PE-NTA₂ multimers tended to exhibit increased background staining oncontrol PBMCs. Background staining was efficiently suppressed byaddition of 0.5% milk powder (as used, e.g., in Western blotting).

SA-PE NTA4 DR4/HA₃₀₆₋₃₁₈ multimers were able to detect moreantigen-specific cells than BSP multimers over a wide range ofconcentrations (FIG. 49). Stimulated PBMCs were incubated for 1 h at 37°C. (A) or room temperature (C) with 10 μg/ml of the indicated HApeptide-loaded multimers and analyzed after washing by flow cytometry.Nonspecific background (control) was determined on TT₆₃₄₋₆₅₃ peptidestimulated PBMCs. Specific multimer staining was assessed in cellsprepared and stained as in (A) over at different concentrations ofmultimers (B). For comparison the frequencies of IFNγ⁺ T cells wasassessed by ICS and flow cytometry with (+) or without (−) peptidestimulation.

Reversibility of Multimer Staining

Biotin-NTA₄, but not BSP multimers, can be rapidly removed from stained,antigen-specific cells (FIG. 50). Peptide-stimulated PBMCs were stainedwith optimal concentrations (28 μg/ml for biotin-NTA₄-SA-PE and 16 μg/mlfor BSP multimer) DR4-HA multimers at 4° C. for 1 h and washed with coldEDTA-free FACS buffer. After the initial acquisition (0 min), imidazole(100 mM final concentration) was added and data acquisitions (10,000CD3⁺CD⁴⁺revents) was performed at 1, 3, 5 and 10 min by flow cytometry.Control cells stained with biotin-NTA₄-SA-PE multimer were not treatedwith imidazole (A). Specific binding observed in (A) was plotted versusthe time elapsed (B). Biotin-NTA₄-SA-PE multimers were observed to bestable in the absence and rapidly reversible in the presence ofimidazole. This prevents multimer staining-induced death ofantigen-specific T-cells, for example, CD4⁺ T cells or CD8⁺ T cells. Asdescribed elsewhere herein.

Comparative Staining of a DR1/ESO₁₁₉₋₁₄₃ Cell Line by DR1/NY-ESO₁₁₉₋₁₄₃BPS, PE-NTA₂ and Biotin-NTA₄-SA-PE Multimers.

The staining reagents were prepared as described above fromDTB/StreptActin purified DR1/NY-ESO₁₁₉₋₁₄₃ monomers. CD4⁺ cells wereincubated with three different multimer dilutions (1:2, 1:4 and 1:8,corresponding to 14 μg/ml, 7 μg/ml, and 3.5 μg/ml for the SA-PE NTA₄ andthe BSP multimers, and to 7 μg/ml, 3.5 μg/ml and 1.75 μg/ml for the PENTA₂ multimers (FIG. 51 white histograms). Mouse splenocytes (C57BL/6J)were used as a negative control (black histograms). The DR1/ESO₁₁₉₋₁₄₃cell line was derived from an HLA-DR1 transgenic mouse immunized withNY-ESO₁₁₉₋₁₄₃ peptide, and was maintained in IMDM-10, 50 μMbeta-mercaptoethanol, pen/strep, with 50 U/ml of rmIL-2.

Example 6 Additional Tags

Additional tags for purification of peptide-loaded MHC monomers wereevaluated (FIG. 52). Earlier elution on anion-exchange chromatography ofpY-E8-HA peptide loaded than DR4-x (x=no/any peptide) from anionexchange chromatography column (mono-Q) upon elution with a lineargradient of NaCl is shown in FIG. 52. The gradient shown as a light linerises from 0 to 1 M NaCl. FIG. 52 B shows an ELISA of DR4 (light line)and pY (phospho-tyrosine)(dark line).

Additional acidic tags that were successfully employed for the isolationof correctly peptide loaded MHC II monomers includes YP-D4-SGSG-*-HA,YS-D6-SGSG-*-HA, YP-E8-GSG-HA, YP-E8-GSG-HA, YP-DDGGDDGGDD-SGS-HA, P—PO₄²⁻, —S—SO³⁻.

Of the tags tested, the pY-E8 tag provided the best results. Whileanionic tags are suitable for the isolation of peptide-loaded MHCmonomers, the disadvantages of using such tags include: i) difficultiesin peptide synthesis, ii) the high NaCl concentration needed to elutethe complexes may partially denature them; iii) an additional desaltingstep is need to remove the high NaCl concentrations.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an”, as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B”, when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of”, when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently, “at least one of A and/or B”)can refer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one act,the order of the acts of the method is not necessarily limited to theorder in which the acts of the method are recited.

1.-12. (canceled)
 13. A protein multimer, comprising (a) a multivalentcarrier molecule, and (b) a plurality of MHC class II molecules bound tothe carrier molecule, wherein at least one of the MHC class II moleculesis conjugated to the carrier molecule via a chelate complex bond,wherein the chelate complex bond comprises a chelant conjugated to atleast one of the MHC class II molecule, and a chelant conjugated to thecarrier molecule, and optionally wherein the chelant conjugated to thecarrier molecule is of a different structure than the chelant conjugatedto the MHC molecule.
 14. The protein multimer of claim 13, wherein thechelate complex bond is a bond with a dissociation constant 5 μM>K_(D)≧1fM. 15.-21. (canceled)
 22. The protein multimer of claim 13, wherein thechelant conjugated to the MHC class II molecule is C-terminallyconjugated to the MHC α chain.
 23. (canceled)
 24. The protein multimerof claim 13, wherein the chelant conjugated to the MHC class II moleculeis a peptide comprising a chelant moiety, and optionally wherein thepeptide comprising a chelant moiety is fused to a polypeptide chaincomprised by the MHC molecule.
 25. (canceled)
 26. The protein multimerof claim 24, wherein the peptide comprising a chelant moiety comprises apoly-Histidine sequence, optionally wherein the poly-Histidine sequencecomprises 3-24 His residues. 27.-28. (canceled)
 29. The protein multimerof claim 13, wherein the chelant conjugated to the carrier moleculecomprises an NTA moiety, wherein the NTA moiety is bound to the carriermolecule in mono-NTA, di-NTA, tetra-NTA, or poly-NTA configuration.30.-32. (canceled)
 33. The protein multimer of claim 29, wherein the NTAmoiety is bound to a linker, and optionally wherein the linker comprisesa maleimide moiety, an oxime moiety, or derivatives thereof, andoptionally wherein the linker is between about 9 Å and about 23 Å long.34.-36. (canceled)
 37. The protein multimer of claim 33, wherein thelinker is covalently bound to the carrier molecule.
 38. The proteinmultimer of claim 33, wherein the linker is covalently bound to a ligandof a binding molecule, and wherein the binding molecule is covalentlybound to the carrier molecule, and optionally wherein the ligand isbiotin and the binding molecule is streptavidin.
 39. (canceled)
 40. Theprotein multimer of claim 13, wherein the chelate complex bond furthercomprises a divalent cation, and wherein the divalent cation is an Ni²⁺,Cu²⁺, Zn²⁺, Co²⁺, Cd²⁺, Sr²⁺, Mn²⁺, Fe²⁺, Mg²⁺, Ca²⁺, or Ba²⁺ ion. 41.(canceled)
 42. The protein multimer of claim 13, wherein the carriermolecule is a fluorophore, a phycobilin, phycoerythrin,allophycocyanine, a quantum dot (Qdot), a microsphere, a fluorescentmicrosphere, a magnetic particle, or a nanoparticle.
 43. The proteinmultimer of claim 13, wherein the MHC class II molecule is an empty MHCclass II molecule or a peptide-loaded MHC class II molecule.
 44. Theprotein multimer of claim 43, wherein the peptide-loaded MHC class IImolecule is chosen from the MHC class II molecules disclosed in Table 2.45. (canceled)
 46. The protein multimer of claim 13, wherein the MHCclass II molecule is loaded with an antigenic peptide, and optionallywherein the peptide comprises the sequence GILGFVFTL (SEQ ID NO: 308).47. (canceled)
 48. The protein multimer of claim 13, wherein themultimer is a tetramer.
 49. The protein multimer of claim 46, whereinthe antigenic peptide is conjugated to a tag, and optionally wherein thetag is conjugated to the peptide via a cleavable linker.
 50. (canceled)51. The protein multimer of claim 49, wherein the linker is aphotocleavable linker, optionally wherein the photocleavable linker isan NPβA linker; or wherein the linker is a peptide linker that comprisesan amino acid sequence that can be cleaved by a protease or by achemical; or wherein the tag is an acidic peptide tag selected from apY-D4, pY-D5, pY-D6, pY-D7, pY-D8, pY-D9, pY-D10, pY-E4, pY-E5, pY-E6,pY-E7, pY-E8, pY-E9, or pY-E10; or wherein the tag is a desthiobiotin(DTB) tag. 52.-90. (canceled)
 91. A method, comprising contacting amonomeric chelant moiety-conjugated MHC class II molecule with awater-soluble carrier molecule conjugated to a plurality of chelantmoieties under conditions suitable for formation of a chelate complexbetween the chelant moieties conjugated to the MHC class II molecule andthe chelant moieties conjugated to the carrier molecule.
 92. The methodof claim 91, wherein the chelant moieties conjugated to the carriermolecule are NTA moieties, wherein the NTA moieties are in mono-NTA,di-NTA, tetra-NTA, or poly-NTA configuration, and wherein the NTAmoieties are bound to a linker, optionally wherein the linker is betweenabout 9 Å and about 23 Å long. 93.-97. (canceled)
 98. The method ofclaim 96, wherein the linker is covalently bound to the carriermolecule; or wherein the linker is covalently bound to a ligand of abinding molecule, and wherein the binding molecule is covalently boundto the carrier molecule, optionally wherein the ligand is biotin and thebinding molecule is streptavidin. 99.-100. (canceled)
 101. The method ofclaim 91, wherein the carrier molecule is a quantum dot (Qdot), amagnetic particle, a nanoparticle, PE conjugated to streptavidin, or afluorophore, optionally wherein the fluorophore is a phycobilin,optionally selected from phycoerythrin or allophycocyanine; or whereinthe carrier molecule conjugated to a plurality of chelant moieties isgenerated by incubating a carrier molecule conjugated to a plurality ofbinding molecules with an excess of chelant-conjugated ligand underconditions suitable for the ligand to bind the binding molecule.102.-108. (canceled)
 109. The method of claim 101, wherein the molarratio of carrier:ligand is between 1:2 and 1:10, or is 1:5. 110.(canceled)
 111. The method of claim 101, wherein the incubating isperformed at a temperature between 2-16° C., or at about 4° C. 112.(canceled)
 113. The method of claim 91, wherein the method comprises astep of incubating the carrier molecules contacted with the ligand withNiSO₄.
 114. The method of claim 91, wherein the method comprises a stepof contacting the carrier molecule conjugated to a plurality of chelantmoieties with a molar excess of the MHC molecule conjugated to achelant, and wherein the excess is 2-20 fold or is 10-fold. 115.-119.(canceled)
 120. The method of claim 91, wherein the MHC class IImolecule is loaded with an antigenic peptide. 121-147. (canceled)
 148. Amethod comprising, providing an MHC molecule bound to an antigenic MHCmolecule-binding peptide that is conjugated to a tag via a cleavablelinker, removing the tag from the antigenic peptide, and conjugating achelant moiety to a heavy chain of the MHC molecule, optionally furthercomprising, contacting a multivalent chelant molecule with the MHCmolecule under conditions suitable for the chelant moiety conjugated tothe MHC molecule to form a chelate complex bind with a chelant moiety ofthe multivalent chelant molecule. 149.-178. (canceled)
 179. An isolatedpeptide-loaded MHC class II molecule, comprising an MHC heavy chain, andan antigenic peptide, wherein the peptide is conjugated to a tag for ionexchange chromatography.
 180. (canceled)
 181. The isolatedpeptide-loaded MHC class II molecule of claim 179, wherein the tag is anacidic tag, and wherein the acidic tag is an acidic cyanine dye or apeptide tag selected from a pY-D4, pY-D5, pY-D6, pY-D7, pY-D8, pY-D9,pY-D10 tag, pY-E4, pY-E5, pY-E6, pY-E7, pY-E8, pY-E9, or pY-E10 tag.182.-185. (canceled)
 186. The isolated peptide-loaded MHC class IImolecule of claim 179, wherein the MHC molecule further comprises aheavy chain that is conjugated to a chelant moiety.
 187. The isolatedpeptide-loaded MHC class II molecule of claim 179, wherein the moleculecomprises a combination of a heavy chain and an antigenic peptidedisclosedin Table
 2. 188. The isolated peptide-loaded MHC class IImolecule of claim 179, wherein the tag is conjugated to the peptide viaa cleavable linker, optionally wherein the linker is a photocleavablelinker, optionally wherein the photocleavable linker is an NPβA linker;or wherein the linker is a peptide linker that comprises an amino acidsequence that can be cleaved by a protease or by a chemical. 189.-191.(canceled)
 192. The isolated peptide-loaded MHC class II molecule ofclaim 179, wherein the MHC class II molecule is comprised in an MHCmultimer.
 193. A method, comprising contacting an empty MHC class IImolecule with an antigenic peptide conjugated to a tag under conditionssuitable for the antigenic peptide to bind the MHC class II molecule.194. (canceled)
 195. The method of claim 193, wherein the tag conjugatedto the MHC class II binding antigenic peptide is an affinity tag that isnot a polyhistidine tag.
 196. The method of claim 193, wherein the tagis an acidic tag, wherein the tag is an acidic cyanine dye, or a peptidetag selected from a pY-D4, pY-D5, pY-D6, pY-D7, pY-D8, pY-D9, pY-D10pY-E4, pY-E5, pY-E6, pY-E7, pY-E8, pY-E9, or pY-E10 tag. 197.-200.(canceled)
 201. The method of claim 193, wherein the tag is conjugatedto the peptide via a cleavable linker.
 202. The method of claim 201wherein the linker is a photocleavable linker, and optionally whereinthe photocleavable linker is an NPβA linker; or wherein the linker is apeptide linker that comprises an amino acid sequence that can be cleavedby a protease or by a chemical; or wherein the tag is a part of acleavable linker that remains after cleavage of the linker. 203.-226.(canceled)